Functional imaging and targeted drug delivery in mice and patient tumors with a cell nucleolus-localizing and tumor-targeting peptide

doi:10.21203/rs.3.rs-1567587/v1

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Research Article

Functional imaging and targeted drug delivery in mice and patient tumors with a cell nucleolus-localizing and tumor-targeting peptide

https://doi.org/10.21203/rs.3.rs-1567587/v1

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posted 21 Apr, 2022

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Tumor-targeting peptides have profound clinical implications in early detection and delineation of microscopic lesions for surgical resection, and also delivery of therapeutics with reduced systemic toxicity. Here, we demonstrate that a peptide (RS), selected from a rationally-designed variant library of a reported hepatocellular carcinoma (HCC)-targeting peptide P47, enables discrimination of HCC from noncancerous tissues in orthotopic HCC mice and patient biopsies, and delineation of micrometastases in lung, with up to 21-fold contrast between tumor and surrounding liver, significantly higher than P47. Importantly, RS targets non-small cell lung (NSCLC) and colon cancers in mice and patient biopsies, with higher selectivity for highly proliferative tumor nodules. Moreover, RS localizes to cell nucleoli of HCC, NSCLC, breast, colon and cervical cancer cells and induces cell nucleolar stress when conjugated with chemotherapeutic Oxaliplatin (OXA) (RS-OXA), demonstrating both cellular and subcellular targeting. RS-delivered OXA elicits significant tumor retardation in orthotopic HCC mice with markedly reduced systemic toxicity compared to OXA alone. Injection of fluorescence-labeled RS enables dynamic visualization of tumor growth in RS-OXA-treated subcutaneous HCC mice. Our study demonstrates that RS targets a spectrum of tumors and localizes to cell nucleolus, and enables functional imaging and targeted delivery of OXA in HCC mice, and thus provides a versatile tool for tumor imaging and targeted therapeutics.

Tumor

HCC

tumor-targeting and cell nucleolus-localizing peptide

chemotherapy

Tumor-targeting peptides show potential in tumor imaging and targeted therapy due to low molecular weight, small size, high affinity and selectivity, low toxicity and production cost.[1, 2] A myriad of tumor-targeting peptides have been identified and extensively tested as molecular probes for tumor imaging or surgical navigation and cancer treatment in preclinical studies.[3–6] Examples include an internalizing-RGD peptide that specifically targets glioblastoma, melanoma, lung, prostate and breast cancer by binding to αvβ3 integrin on tumor cell surfaces.[7]^, [8, 9] NGR that targets CD13 on endothelial cells of colorectal, ovarian, non-small-cell lung cancer (NSCLC), small-cell lung cancer (SCLC) and hepatocellular carcinoma (HCC)[10, 11] has been tested in clinical trials.[12] However, none of them shows the ability to enter tumor cell nucleoli.

As a non-membrane-bound sub-nuclear structure and a factory for ribosome biogenesis, [13]^, [14] the cell nucleolus represents a promising subcellular therapeutic location as hyperactive ribosome biogenesis was shown to contribute to aggressiveness and resistance of cancers.[15] Probes tracking cell nucleolus have been extensively explored but most of them lack tumor-targeting property.[16, 17] Moreover, multidrug resistance (MDR) induced by ATP-binding cassette (ABC) transporters on plasma membrane and systemic toxicity are the major hurdles for chemotherapeutic treatment of cancers,[18, 19] specific delivery of chemotherapeutic drugs to tumor and cell nucleolus can avoid these issues. Therefore, approaches to target tumor and cell nucleolus have the potential to unlock the full potential of tumor chemotherapy with manageable safety risks, particularly in difficult-to-treat tumors such as HCC.

HCC is highly refractory to conventional chemotherapy,[20] due to its high pathological heterogeneity and high metabolic activity.[21] Several oxaliplatin (OXA)-based regimens resulted in improved response rates and survival benefits compared to doxorubicin and other platinum-based chemotherapeutic drugs in clinical trials for advanced HCC;[22] however OXA therapy suffered from severe drug-related adverse effects including systemic toxicity and MDR after repeated doses.[23, 24] Moreover, OXA was shown to effect nucleolar or ribosome biogenesis stress rather than trigger DNA damage response, unlike other platinum-containing chemotherapeutic agents.[25, 26] Thus, tumor- and cell nucleolus-targeted delivery of OXA is critical for antitumor efficacy on tumor cells.

Previously, we identified a HCC-targeting peptide (P47) that enabled detection of tumor micronodules in mice and patients with HCC; [27] however, it showed limited effect on other tumors and was unable to enter cell nuclei. To improve its ability to target a large spectrum of tumors and cell nuclei, particularly, cell nucleolus, we rationally designed 384 peptide variants based on P47 sequence with a bioinformatics tool (http://crdd.osdd.net/raghava/tumorhpd). Among predicted peptide variants, RS outperformed P47 and other variants and showed greater specificity for HCC, NSCLC and colon cancers over peritumoral tissues in mice and patient biopsies, and improved targeting ability to NSCLC, breast, colon and cervical cancers in mice. Importantly, RS effectively localized to cell nucleolus of HCC, NSCLC, breast, cervical and colon cancers, a property was not observed with P47. RS directed HCC-targeted delivery and cell nucleolus localization of OXA in vitro and in orthotopic HCC mice when conjugated with OXA (RS-OXA), with significantly reduced systemic toxicity. Dynamic monitoring of tumor growth was achieved when fluorescence-labeled RS were simultaneously injected in RS-OXA-treated subcutaneous HCC mice. Our study demonstrates that RS enables clear discrimination of a variety of tumors from peritumoral tissues in mice and patients and directs tumor- and cell nucleolus-specific delivery of OXA in HCC mice, and thus provides a new tool for tumor imaging and targeted therapy.

Rationale design identifies lead peptide with strong HCC-targeting ability

To enhance the tumor-targeting and cell-permeabilizing ability of P47,[27] we employed a bioinformatics tool (http://crdd.osdd.net/raghava/tumorhpd/ and https://webs.iiitd. edu.in/raghava/tumorhpd/), a TumorHPD web server for tumor-homing peptide prediction,[28] to identify P47 variants with stronger tumor-targeting and cell- permeabilizing ability based on their support vector machine (SVM) scores, hydrophilicity or solubility, charge and isoelectric point (pI).[29] To determine which end is more important for its targeting ability, we coupled FAM to different ends of P47 and administered the peptides to subcutaneous HCC mice. Weaker fluorescence signals were detected in tumor with FAM coupled to the C terminus than the N terminus (Figure S1a), indicating that the C terminus may be more important. Thus, mutations were largely introduced to the N terminus of P47. Systemic intravenous injection of top two variants with similar SVM scores revealed significantly stronger fluorescence signals with FAM-labeled V2 than V1 and P47 peptides in tumor of subcutaneous HCC mice under identical conditions (Figure 1a-c). Based on the strong tumor-targeting efficacy of V2, we generated more V2 mutants. Strikingly, more specific tumor accumulation was observed with V2-RS in subcutaneous HCC mice (tumor-liver ratio of 28.37±0.80) than P47 (8.24±0.80) (Figure 1b and 1c), demonstrating a stronger HCC-targeting potency of V2-RS (RS). Consistently, although V2-RK, -RA, or -RL showed slightly higher SVM scores than RS, there was no further improvement compared to parental V2 (Figure 1b and 1c).

To investigate RS in a more clinically relevant setting, we intravenously administered FAM-labeled RS and P47 in orthotopic HCC mice at the dose of 25 mg/kg for single injection. Although both RS and P47 were able to clearly delineate the margin between tumor and surrounding tissues, RS showed stronger fluorescence signals and higher tumor-liver contrast (21.22 ± 0.97) than P47 (7.56 ± 0.36) (Figure 1d), a difference similar to that of subcutaneous HCC mice. As micrometastases largely account for post-surgical recurrence, [30] we examined the effect of RS on lung micrometastasis in HCC mice in by intravenously injected Hepa1-6 cells to wild-type mice.[31] Remarkably, RS labeled metastatic HCC micronodules of 0.1mm in diameter in lungs of mice (Figure 1e). In contrast, P47 was only able to mark micronodules with a diameter of 0.3mm in HCC mice bearing metastatic mCherry-expressing tumors in lungs (Figure S1b). As detection of deeply-located HCC nodules is typically challenging during surgery, [32] to investigate the feasibility of visualizing tumor nodules deep inside the liver, we labeled RS with quantum dot (Ag₂S-RS) and intravenously injected Ag₂S-RS in orthotopic HCC nude mice bearing human HCC tumors at the dose of 4mg/kg. Both deeply-located and superficial tumor micronodules were clearly highlighted with Ag₂S-RS (Figure 1f), confirming the sensitivity and specificity of RS. Notably, RS showed high serum stability than P47, [27] with 71.8% of the peptide remained intact in 20% serum at 8 h, as revealed by liquid chromatography-electrospray mass spectrometric (LC-MS) analysis (Figure 1g). Similar to P47, [27] RS was also sequentially degraded from the C terminus (Figure 1g). Moreover, intravenous injection of RS at high dose (100mg/kg) did not induce any detectable toxicity in wild-type mice reflected by levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (Figure S1c) and normal morphological structures of liver and kidney (Figure S1d). Altogether, these findings demonstrated that RS targets HCC with high sensitivity, specificity and serum stability.

RS discriminates tumor from peritumoral tissue in HCC and ICC patient biopsies

To investigate the clinical applicability of RS in differentiating tumor from abnormal liver in HCC patients, we applied FAM-labeled RS to surgically excised fresh tumor and peritumoral liver tissues. Clear delineation of tumor margins or microscopic lesions from surrounding tissues was achieved in HCC patient biopsies with FAM- labeled RS, with microscopic lesions of 0.1mm in diameter (Figure 2a) and residual tumor tissues after surgery being highlighted (Figure S2). RS was also able to detect tumor infiltration in the liver of HCC patients (Figure 2b), a phenomenon frequently observed in HCC patients.[33] Importantly, RS enabled the discrimination of HCC tumors with high or low proliferative activities, characterized by the abundance of ribosomes and size of nucleoli,[34] with substantial accumulation in highly proliferative tumor nodules (Figure 2c), implying that RS might target with efficacy proportional to tumor aggressiveness. Consistently, a much weaker fluorescence signal was found in hemorrhagic necrosis areas of HCC tissues with low proliferative activity, manifested by karyorrhexis and karyolysis,[35] than other areas in HCC patient biopsies (Figure 2d). Strikingly, RS demonstrated clear differentiation of tumor from highly heterogeneous surrounding liver tissues including normal-like liver, lipid degeneration, inflammation, fibrosis, cirrhosis and nodular hyperplasia when HCC patient biopsies were assembled in order of disease progression, confirmed by histological analysis (Figure 2e), strengthening the therapeutic potential and general applicability of RS in HCC patients at different stages. Moreover, RS discriminated intrahepatic cholangiocarcinoma (ICC) with proliferative bile ducts from peritumoral liver tissues with severe inflammatory infiltration in patient biopsies (Figure 2f). Clear differentiation of ICC and HCC in the combined ICC-HCC patient cases was observed in a highly heterogeneous background including moderate lipid degeneration, inflammatory infiltration and bile duct proliferation (Figure 2g), with a comparable tumor-liver ratio between ICC and HCC (Figure 2h), demonstrating the clinical applicability of RS in detecting HCC and ICC.

RS targets different tumors in mice and patient biopsies

To examine whether RS is able to target other tumors besides HCC, we intravenously administered FAM-labeled RS into nude mice bearing subcutaneous human non-small cell lung (NSCLC), breast, cervical and colon tumors. Strikingly, RS specifically accumulated in tumors of different types, with HCC ranking top and NSCLC and colon cancer secondary to HCC based on the ratio of tumor relative to corresponding tissues (Figure 3a and 3b). Unsurprisingly, a clear delineation of tumor from peritumoral lung tissues was achieved in mice bearing orthotopic murine and human NSCLC, with a tumor/ lung ratio of 6.31±1.12 and 8.24±0.49, respectively (Figure 3c). Correspondingly, RS clearly differentiated lung adenocarcinoma from peritumoral lungs with infiltrating monocytes, pneumoconiosis and fibrosis in patient biopsies (Figure 3d and 3e). Importantly, RS enabled marked discrimination of highly proliferative lung adenocarcinoma, characterized by papillary or micropapillary structures and high nucleo-cytoplasmic ratio,[36] from less proliferative tumors, reflected by the appearance of simple acinar structures,[37] in patient biopsies, with a significantly greater tumor/ lung ratio in highly than less proliferative lung adenocarcinoma (Figure 3e), including the hemorrhagic necrosis area of lung adenocarcinoma tissues (Figure 3f). A similar pattern was observed in lung squamous carcinoma, with RS showing stronger fluorescence signals in highly proliferative, represented by clustered tumor cells with high nucleo-cytoplasmic ratio[38], than poorly proliferative tumors (Figure 3g), characterized by the formation of keratin pearl at the center of cancer nest.[39] Similarly, RS also distinguished colon adenocarcinoma from peritumoral colon tissues in patient samples with similar indications as NSCLC and HCC (Figure S3). These data showed that RS targets a broad spectrum of tumors with selectivity for highly proliferative tumor nodules.

RS specifically localizes to tumor cell nucleolus

Considering RS’s broad tumor-targeting ability, we wondered whether RS can be readily internalized by different tumor cells. Substantially stronger fluorescence signals were observed in human HCC cells treated with FAM-labeled RS than P47, with faint signals found in noncancerous human hepatocytes (Figure 4a and 4b). As expected, significantly greater cellular uptake was yielded in human NSCLC, colon, breast and cervical cancer cells treated with FAM-labeled RS, whereas negligible levels were detected with P47 under identical conditions (Figure 4a-c), indicating the potent tumor cell internalization capacity of RS. Strikingly, stronger fluorescence signals were found in cell nuclei, with punctate foci in cell nucleoli, of human HCC cells treated with FAM-labeled RS. P47 predominantly localized in cytoplasm with small amounts scattered in nuclei[27] (Figure 4d). Similar observations were detected in other human HCC and tumor cells, with substantial amounts of RS accumulated in cell nucleoli (Figure 4e), confirming the strong tumor cell nucleolus-localizing capacity of RS.

RS augments OXA’s specific uptake and cytotoxicity in HCC cells in vitro

RS’s tumor-targeting and cell nucleolus-localizing properties could be therapeutically useful, thus we assessed RS-mediated delivery of OXA, a first-line chemotherapeutic agent for HCC patients with primary effects in cell nucleolus.[25] RS-OXA with 96.43% purity and molecular weight of 1928.84 was synthesized (Figure S4a and S4b). Remarkably, strong fluorescence signals were observed in tumor cell nucleolus when AF680-labeled RS-OXA was incubated with human HCC cells, but not in noncancerous human hepatocytes (Figure 5a), indicating tumor cell nucleolus-specific uptake of OXA. As OXA primarily induces nucleolar stress,[25, 26] we examined the translocation of nucleophosmin (NPM1), an indicator for nucleolar stress characterized by translocation from cell nucleoli to nucleoplasm in response to stress,[26] in human HCC cells. As expected, NPM1 was largely diffused in cell nucleoplasm of HCC cells treated with RS-OXA, whereas the expression of NPM1 appeared as punctate foci in cell nucleoli of PBS- or OXA-treated HCC cells (Figure 5b). In clear contrast, no evident NPM1 expression was detected in nucleoplasm of noncancerous hepatocytes treated with either RS-OXA or OXA (Figure 5b), demonstrating that RS promotes HCC- and cell nucleolus-specific uptake of OXA and results in nucleolar stress. Correspondingly, significantly higher rates of cellular apoptosis, reflected by annexin V and propidium iodide (PI) double staining [40] (Figure 5c and 5d), and cytotoxicity (Figure 5e) were triggered by RS-OXA than by OXA in human HCC cells. Notably, RS-OXA had a much less impact on human noncancerous hepatocytes than OXA (Figure 5c-e). Unsurprisingly, human HCC cell invasion ability was also dramatically compromised by RS-OXA, to a much less extent with OXA, whereas no migratory inhibition was observed with RS-OXA-treated human noncancerous hepatocytes (Figure 5f). However, OXA induced a significant inhibitory effect on cell migration in human noncancerous hepatocytes (Figure 5f), indicating the nonspecific cell toxicity. These results demonstrated that RS augments OXA’s tumor- and cell nucleolus-specific uptake, resulting in cell nucleolar stress, cytotoxicity and migratory inhibition in vitro.

RS enables tumor detection and specific OXA delivery in HCC mice

To investigate the ability of RS to direct tumor-specific delivery of OXA in vivo, we intravenously administered RS-OXA into day-7 subcutaneous HCC mice at the dose of 10 μmol/kg for 5 times every 3 days apart (Figure 6a), a dosing regimen adopted from previous studies.[41] As real-time monitoring of the therapeutic effect of drugs is critical for clinical assessment, we injected AF680-labeled RS intravenously at the dose of 500 μg/kg at different time-points and examined mice 12 h later (Figure 6a). RS-OXA significantly inhibited tumor growth compared to OXA alone under identical conditions (Figure 6b and 6c). Importantly, AF680-labeled RS enabled dynamic visualization and clear delineation of tumors in pretreated and treated tumor- bearing mice (Figure 6b and 6c), indicating the potential of RS for targeted therapeutic intervention and prognostic monitoring. Consistently, RS-OXA triggered sustainable tumor suppression in day-7 orthotopic HCC mice under identical dosing conditions (Figure 6d), reflected by significant tumor retardation in RS-OXA-treated mice compared to OXA-treated and untreated mice (Figure 6e and 6f), demonstrating the specific antitumor potency of RS-OXA. Strikingly, RS-OXA also prevented pulmonary metastasis in orthotopic HCC mice, whereas prominent metastasis was found in mice treated with OXA and untreated controls (Figure 6g). Notably, no morphological changes were observed in kidneys across different treatment groups (Figure S4c), which is consistent with previous reports on OXA.[42] Corroborating with negligible adverse effects observed with RS- OXA, significantly higher amounts of Pt accumulated in tumor of mice treated with RS-OXA than OXA, whereas negligible in surrounding liver tissues (Figure 6h). In contrast, comparable levels of Pt were found in tumor and liver tissues of mice treated with OXA (Figure 6h). Importantly, RS-OXA did not evoke any acute neuropathy, an adverse effect frequently occurred with OXA treatment,[43] reflected by significantly less neuropathic pain in nude mice treated with RS-OXA than OXA and comparable to untreated controls when exposed to cold (Figure 6i). Also sinusoidal obstruction syndrome (SOS), a liver parenchymal damage primarily involving sinusoids, [44] was markedly reduced in liver of nude mice treated with RS-OXA compared to OXA (Figure 6j). Moreover, tumor-targeted delivery of OXA reduced its adverse effect on bodyweight loss of nude mice (Figure S4d). Altogether, these findings confirmed that RS directs tumor-targeted delivery of OXA, and resulting in sustainable tumor inhibition with improved safety profiles.

One of major goals for molecular imaging is the ability to characterize microscopic lesions in vivo to avoid the added risks associated with the removal of benign surrounding tissues.[44] Likewise, targeted delivery of chemotherapeutic agents to malignant cells, particularly to cell nucleolus, is able to minimize systemic toxicity and potential drug resistance related to ATP-binding cassette (ABC) transporters on plasma membrane.[15] Here, we identified a peptide (RS) that enables clear delineation of tumor from peritumoral tissues in mice and patient biopsies of HCC, ICC, NSCLC and colon cancers, with higher selectivity for actively proliferating tumor cells in patient biopsies. Importantly, RS specifically localized to cell nucleolus and mediated tumor- and cell nucleolus-specific delivery of OXA, resulting in tumor cell nucleolar stress and cytotoxicity in vitro and significant tumor suppression in HCC mice, with markedly reduced adverse effects and systemic toxicity. Injection of fluorescence- labeled RS enabled dynamic monitoring of tumor growth and drug efficacy in HCC mice treated with RS-OXA. Our study provides a new tool for functional imaging and targeted drug delivery in a wide array of cancers.

Although OXA has been extensively used a front-line treatment for different cancers, particularly for HCC, [22] one major hurdle for the clinical implementation of OXA is systemic toxicity.[43] In our current study, we tagged OXA with RS and achieved tumor- and cell nucleolus-specific delivery of OXA, yielding augmented therapeutic efficacy with minimal systemic toxicity in HCC mice. Worthy mentioning, RS can be routinely synthesized and scaled up at low costs, and conjugation with OXA does not require sophisticated chemical techniques, thus it is likely that RS-OXA can be manufactured easily and fast-tracked to the clinic upon completion of extensive toxicological and pharmacokinetic studies of RS. Although we did not detect any liver or kidney toxicity in mice at tested doses, safety studies in large animals at higher doses are essential prior to clinical deployment of RS. In addition, multi-drug resistance elicited by ATP-binding cassette (ABC) transporters on plasma membrane is another critical issue for chemotherapy,[18] RS-mediated cell nucleolus-specific drug delivery can potentially overcome this and extensive studies on this aspect are warranted in future.

Post-surgical recurrence is a common phenomenon in tumor including HCC, lung and colon and other cancers.[45–47] Among them, HCC ranks top with 70.2% recurrence rate within 84.4 months after surgery.[48] Given high recurrence rates are closely associated with the failure to detect micrometastases with current imaging techniques; [49] there is a clear need to identify molecular probes with higher sensitivity and specificity. The ability of RS to detect microscopic lesions with a diameter of 0.1mm would greatly enhance detection of micrometastases and facilitate surgical navigation and reduce chances of surgical recurrence, potentially increasing 5-year survival rates of HCC, by extension to other cancer patients. Importantly, we demonstrated that RS can differentiate tumor nodules of different proliferative activities, with higher selectivity for highly proliferative tumor nodules. This unique feature can be exploited to target the remaining post-surgical micrometastases, which are largely highly proliferative and the main contributors to the recurrence.[30] By incorporating RS to imaging tracers of positron emission tomography (PET) to pinpoint microscopic lesions with high proliferative activities, RS can be used to guide local radiotherapy and other interventions and also to predict tumoral response. Therefore, our findings may have significant implications for surgical navigation or post-surgical monitoring and PET- guided targeted therapy.

In summary, we demonstrated that RS targets a broad spectrum of tumors with high sensitivity and specificity and localizes to cell nucleolus and RS-mediated tumor- and cell nucleolus-specific delivery of OXA significantly enhances OXA’s activity with minimal systemic toxicity in HCC mice, thus providing a new tool for functional imaging and targeted drug delivery.

In summary, we identified a peptide (RS), which targets a spectrum of tumors and localizes to cell nucleolus and enables functional imaging and targeted delivery of OXA in HCC mice, and thus provides a versatile tool for tumor imaging and targeted therapeutics in a variety of tumors.

Animals and injections

C57BL/6 wild-type and BALB/C nude mice (6-8-week old) were used in all experiments (the number used was specified in Figure legends). Mice were housed under specific pathogen free conditions in a temperature-controlled room. The experiments were carried out in the Animal unit, Tianjin Medical University (Tianjin, China), according to procedures authorized by the institutional ethical committee (Permit Number: SYXK 2019-0004). Mice were sacrificed by CO₂ inhalation or cervical dislocation at desired time-points, and tissues were fixed with Bouin’s solution (Sigma, St. Louis, MO, US) and embedded with paraffin for immunohistochemical and histological studies.

OXA or RS-OXA was dissolved in N,N-dimethylformamide (DMF)/ Tween 80/ PBS (5:1:94, volumetric ratio) and injected intravenously at the dose of 10μmol /kg into mice bearing subcutaneous or orthotopic HCC tumors for 5 times every 3 days apart. The length (L) and width (W) of subcutaneous HCC tumor were measured with a caliper at 7, 10, 14, 17 and 21 days after tumor implantation. Tumor size was calculated by the formula: (L×W²)/2. While tumor size of orthotopic HCC tumor was calculated based on MRI images at 1, 2, and 3 weeks after tumor implantation. For real-time monitoring of the therapeutic effect of RS-OXA, AF680-labeled RS peptides were intravenously injected into subcutaneous HCC mice 12 h prior to imaging with the IVIS spectrum imaging system (PE, Waltham, MA, US).

Clinical samples

The hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), combined HCC and ICC, colon adenocarcinoma (CA) and NSCLC patient biopsies used in this study were provided by Tianjin Medical University General Hospital and Cancer Hospital (Permit Number: bc2020175). All patients provided written informed consent for the sample collection. This study was conducted in accordance with Declaration of Helsinki and was approved by the Tianjin Medical University Cancer Hospital Ethics Committee.

Establishment of tumor-bearing mouse models

To establish mouse models bearing subcutaneous tumors, MHCC-LM3 (5×10⁵), Hepa1-6 (1×10⁶), A549 (1×10⁶), HCT116 (1×10⁶), MDA-MB-231 (1×10⁶) and Hela (1×10⁶) cells were suspended in 70 μl PBS and injected into C57BL/6 or BALB/C nude mice subcutaneously. For orthotopic HCC mice, subcutaneous tumors with a longitudinal diameter of 1cm were collected from subcutaneous mice after CO₂ inhalation or cervical dislocation. Tumor tissues were washed in D-hanks buffer and necrotic tumor tissues were removed and remaining tumor tissues were cut into about 1mm³ pieces. The recipient mice were anesthetized with isoflurane and skin was sterilized with iodophor 3 times before surgery as described previously.[31] Tumor tissues (3-5 pieces) were implanted in the left lobe of liver in the recipient mice under anesthesia, and the peritoneum and skin were closed with 4-0 sutures. For HCC mice bearing lung metastasis, 1×10⁶ MHCC-LM3 cells were suspended in 100 μl PBS and intravenously injected into BALB/C nude mice within 5 seconds. For mice bearing orthotopic murine and human NSCLC, LLC1 (1×10⁶) or A549 (1×10⁶) cells were suspended in 100 μl PBS and intravenously injected into C57BL/6 or BALB/C nude mice within 5 seconds.

Cellular uptake

Peptides were synthesized and purchased from China Peptides (Shanghai, China) with purity higher than 95%. HL7702, MHCC-97H, Hep3B, MHCC-LM3, HCT116, A549, MDA-MB-231 and Hela cells (10⁴ cells/well) were cultured in serum-free medium for 2 h prior to the addition of FAM-labeled P47 or RS peptides (2 μM) and incubated for another 6h. Cells were washed with PBS for 5 times and observed with fluorescence microscope (IX70, Olympus, Tokyo, Japan). Flow cytometry (BD FACS Calibur, Franklin Lakes, NJ, US) was used for measuring cellular uptake and data were analyzed with the software FlowJo (Tree Star Inc., Ashland, OR, US). To examine the co-localization, FAM-labeled P47 or RS-treated cells were fixed with 4% PFA and counter-stained with DAPI for 15 minutes at room temperature (RT), followed by observation with a confocal laser scanning microscope (LSM700, Carl Zeiss, Germany) with a 40× objective lens.

Serum stability assay

The RS peptide (1 mM) was mixed with 20% mouse serum at a volume ratio of 4:1 and incubated at 37°C for different time-points (0 minute, 1 h, 2 h, 4 h or 8 h). 100 mM dithiothreitol (DTT) was added to reduce the formation of disulfide bonds. Trichloroacetic acid (TCA, 10%) was used to precipitate serum proteins. Subsequently, the solution was cooled at -20°C for at least 20 minutes and centrifuged at 6000g for 10 minutes, and then the supernatants were collected for liquid chromatography-mass spectrometry (LC-MS) analysis (Agilent 7500ce, Agilent Technologies, Waldbronn, Germany).

In vitro binding assay

Fresh HCC, ICC, combined HCC and ICC, CA and NSCLC patient biopsies including tumors and peritumoral tissues were incubated with 10 μM FAM-labeled RS peptides for 30 minutes at 37℃, followed by washing with PBS 5 times for 2 minutes each time. Then the tumor and peritumoral tissues were imaged with the IVIS spectrum imaging system (PE, Waltham, MA, US).

Tissue distribution

FAM-labeled peptides were diluted in 150 μl PBS and administered into C57BL/6 or BALB/C nude mice bearing subcutaneous or orthotopic HCC, NSCLC, CA, breast and cervical cancers intravenously at a dose of 25 mg/kg. Treated mice were terminally anesthetized 2 h post-injection and perfused with 50 ml of cold PBS to wash out unbound peptides. Brain, quadriceps, lung, spleen, kidney, heart, liver, tumor and lung metastases were harvested for imaging with IVIS spectrum imaging system (PE, Waltham, MA, US). RS modified Ag₂S quantum dots (RS-Ag₂S) was diluted in 150 μl PBS and intravenously administered into BALB/C nude mice bearing orthotopic HCC at a dose of 10 mg/kg. The liver of treated mice was imaged 6 h after injection with a NIR-II image system developed by Institute of Automation (Chinese Academy of Sciences, Beijing, China). This system has two CCD cameras: an electron- multiplying CCD (EMCCD) was used to collect NIR-II fluorescent images (ProEM, Princeton Instrument, US); another color CCD was used to collect visible color images (Pilot piA1400-17gc, Basler, Germany). Illumination was provided by a light emitting diode (LED) (NIR light source, center wavelength 808 nm, maximum power 20W) for RS-Ag₂S excitation and a 150W halogen lamp (KL1500LCD, SCHOTT, Germany) for white light color imaging.

Immunohistochemistry and histology

RS was diluted in 150 μl PBS and administered into C57BL/6 mice at a dose of 50 mg/kg. Mice were sacrificed 24 h after injection and liver/ kidney were harvested for histological examination. Serum was collected from the jugular vein for analysis of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (Clinical pathology laboratory, Tianjin Metabolic Disease Hospital, Tianjin, China). Routine H&E staining was used to examine the morphology and pathology of liver, lung, kidney, HCC tumor, lung metastases and clinical tissues. Briefly, tissues were fixed in Bouin’s solution (Sigma, St. Louis, MO, US), embedded with paraffin and cut into 8 μm-thick sections. Subsequently, slides were baked at 60°C for 1 h and then taken through xylene and graded ethanols into distilled water, followed by incubation in filtered 0.1% Mayers Hematoxylin for 10 minutes for nucleus staining. And sections were rinsed in cool-running water to remove excessive hematoxylin, and treated with 0.5% hydrochloric acid and sodium bicarbonate solution (1%), respectively, followed by incubation in 0.5% eosin (0.5g dissolved in 100ml of 95% EtOH) for 5 minutes for cytoplasm staining, and then washed by cool-running water. Sections were dehydrated in graded ethanol for 5 minutes each time and then treated in xylene for 10 minutes and mounted with neutral gum for observation.

Magnetic resonance imaging (MRI)

The magnetic resonance images of orthotopic HCC mice were acquired using T2 propeller sequence with the following parameters: slice thickness of 1.0mm, slice spacing of 0.5mm, TR/TE of 3494/70.7ms, matrix of 256x160 and FOV of 8x8 cm (3.0 Tesia MR scanner, Signa Excite HDx; GE healthcare, Milwaukee, WI, US) with a small animal coil in Tianjin Medical University General Hospital. During the examination, mice were anesthetized with pentobarbital sodium and fixed to minimize body motion.

RS-OXA synthesis and Immunocytochemistry

RS-OXA [Pt(1R,2R-DACH)(ox)(suc)(sucRS)] were synthesized by China Peptides (Shanghai, China). For cellular co-localization, AF680-labeled RS-OXA (5 μM) were incubated with HL7702 or MHCC-LM3 cells for 24 h, followed by fixation with 4% PFA for 30 minutes at 4 °C and counterstaining with DAPI for 15 minutes at room temperature (RT). To examine the translocation of nucleophosmin (NPM1), HL7702 or MHCC-LM3 cells were treated with 5 μM RS-OXA or OXA for 24 h, and fixed with 4% PFA for 30 minutes at 4°C, followed by permeabilization with 0.1% Triton X-100 for 30 minutes and blocking with 5% goat serum for 2 h at RT. Subsequently, cells were stained with primary rabbit monoclonal antibody to NPM1 (1:100, Abcam, UK) at 4°C overnight, and then stained with Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (1:200; Molecular Probes, Thermo Scientific, US) in PBS with Tween 20 (PBST) for 1 h at RT, followed by counterstaining with DAPI for 15 minutes at RT. Cells were photographed using a confocal laser scanning microscope (LSM700, Carl Zeiss, Germany).

Annexin V and propidium iodide (PI) staining

OXA- and RS-OXA-induced cell apoptosis was measured with an Annexin V and Propidium Iodide (PI) Apoptosis Analysis Kit (Tianjin Sungene Biotech Co, Tianjin, China) as per manufacturer’s instructions. Briefly, HL7702 or MHCC-LM3 cells (1×10⁶) were cultured in 6-well plates overnight and treated with RS-OXA or OXA (10 μM) at 37°C for 36 h. Subsequently, cells were rinsed twice with cold PBS and trypsinized (without EDTA) for collection. The collected cells were resuspended in 250 μL of 1× binding buffer and stained with Annexin V-FITC in the dark at 25°C for 15 minutes, followed by incubation with 5 μL of 5 μM PI prior to flow cytometry (BD FACS Calibur; BD, Franklin Lakes, NJ, US).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

MTT assay was applied to determine the cytotoxic effect of RS-OXA in vitro.[50] Briefly, HL7702 and MHCC-LM3 cells were cultured in 96-well plates (2000 cells per well) for 24 h prior to treatment with OXA or RS-OXA at different concentrations (5, 10, 20, 40 and 80 μM) for 48 h. Subsequently, 10 μL of MTT (5 mg/mL in PBS) was added to each well for 4 h, and then the medium was replaced with DMSO (100 μL) for measurement. The samples were measured at 570nm using an enzyme-linked immunosorbent assay (ELISA) reader (Tecan, Sunrise, Switzerland).

Transwell assay

Transwell assays were performed to determine tumor cell invasion with Millipore Transwell chambers (8 μm pore size, Millipore, MA, US) coated with 1 mg/ml Matrigel (Corning, US). HL7702 and MHCC-LM3 cells were treated with 5 μM OXA or RS-OXA for 24 h prior to seeding in the upper chamber of a 12-well plate (3×10⁴ per well) (Corning, US) in 500 μl serum-free medium and the lower chambers were filled with 1 ml complete medium and incubated at 37 °C for 24 h. Subsequently, cells at the upper surface of the membrane were removed with a cotton swab and the cells in the lower surface of the membrane were stained with 1% crystal violet for 15 minutes (Solarbio, China). Images were taken with an inverted microscope (Olympus BX51, Japan) and analyzed using ImageJ software (NIH, Bethesda, US).

Behavioral and toxicity tests

BALB/C nude mice were treated with PBS, OXA or RS-OXA (10 μmol/kg) for 5 times every 3 days apart. Bodyweight was recorded every two days after treatment. Events related to pain were recorded 24 h after the 5^th injection. Mice were placed on the Cold Plate (model 35100; Ugo Basile, Italy) at 4°C, and were free to move. The number of pain signs including rear licking and shaking, jumping, alterations in rear and tail movements was recorded consecutively for 5 minutes. After the cold-elicited behavioral tests, mice were sacrificed by cervical dislocation and liver/ kidney were harvested for histological examination.

Measurement of Pt in tissues

Tumor and liver tissues of orthotopic HCC mice were lyophilized and grinded with each 0.1 g tissue digested with HNO₃ (65%) at 65°C for 10 minutes, and the level of Pt in tissues were measured by Inductively coupled plasma-mass spectrometry (ICP-MS) (Optima 5300DV, PerkinElmer, US).

Statistical analysis

All data are reported as mean values± SEM. Statistical differences between different groups were evaluated by SigmaStat (SystatSoftware, Inc, USA) with significance set at p<0.05. Both parametric and nonparametric analyses were applied as specified in Figure legends. Sample size was determined by PASS software (version 11; NCSS, UT, US).

NSCLC

Non-small-cell lung cancer

SCLC

Small-cell lung cancer

HCC

Hepatocellular carcinoma

MDR

multidrug resistance

ABC

ATP-binding cassette

isoelectric point

LC-MS

liquid chromatography-electrospray mass spectrometric

AST

aspartate aminotransferase

ALT

alanine aminotransferase

ICC

intrahepatic cholangiocarcinoma

NPM1

nucleophosmin

SOS

sinusoidal obstruction syndrome

PET

positron emission tomography

DMF

N,N-dimethylformamide

colon adenocarcinoma

RS-OXA

[Pt(1R,2R-DACH)(ox)(suc)(sucRS)]

PBST

PBS with Tween 20

Ethical Approval and Consent to participate

The mice experiments were carried out in the Animal unit, Tianjin Medical University (Tianjin, China), according to procedures authorized by the institutional ethical committee (Permit Number: SYXK 2019-0004).

The hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), combined HCC and ICC, colon adenocarcinoma (CA) and NSCLC patient biopsies used in this study were provided by Tianjin Medical University General Hospital and Cancer Hospital (Permit Number: bc2020175). The study was conducted in accordance with Declaration of Helsinki and was approved by the Tianjin Medical University Cancer Hospital Ethics Committee.

Consent for publication

All authors are content with and have agreed to submission of this manuscript to Molecular Cancer for publication.

Availability of supporting data

All data supporting this study are available within this article and supplementary Information file.

Competing interests

H.Y., R.J. and R.L. file a patent (202110167783.2). The authors declare no competing financial interests.

Funding

This study was supported by National Natural Science Foundation of China (no. 82030054), Natural Science Foundation of Tianjin (20JCQNJC00580), and Tianjin Municipal 13th five-year plan (Tianjin Medical University Talent Project).

Authors' contributions

H.Y. designed the project; R.J., Q.W., R.L., L.Z., X.Q., J.X., and B.Z. carried out the experiments; L.C., G.L., H.Z, J.C. and T.S helped with clinical sample collection; Y.S. helped with in vitro patient sample experiments; R.J. and H.Y. analyzed the data; H.Y. and R.J wrote the paper with the input from all authors.

Acknowledgements

We thank Core facility of Research Center of Basic Medical Sciences (Tianjin Medical University) for technical support, specifically the animal imaging core facilities.

Authors' information

Renwei Jing and Qian Wang contributed equally to this work.

Affiliations

The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics & Department of Cell Biology & School of Medical Technology, Tianjin Medical University, Qixiangtai Road, Heping District, Tianjin, 300070, China

Renwei Jing, Qian Wang, Ruibin Li, Leijie Zhang, Bingfeng Zuo, HaiFang Yin.

Department of Genetics & Cancer Institute and Hospital, Tianjin Medical University, Tianjin 300070, China

Lu Chen, Guangtao Li, Tianqiang Song.

Department of Lung Cancer Surgery, Tianjin Medical University General Hospital, Tianjin 300070, China

Hongbing Zhang, Jun Chen.

Institute of Bioengineering and Bioimaging, 31 Biopolis Way, 138669, Singapore

Yiqi Seow.

Institute of Molecular and Cell Biology, 61 Biopolis Way, 138668, Singapore.

Yiqi Seow.

Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University, Qixiangtai Road, Heping District, Tianjin, 300070, China.

Xin Qiao, Jingyuan Xu.

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Competing interest reported. H.Y., R.J. and R.L. file a patent (202110167783.2). The authors declare no competing financial interests.

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Functional imaging and targeted drug delivery in mice and patient tumors with a cell nucleolus-localizing and tumor-targeting peptide

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