ChiTn, a mouse/human chimeric anti-Tn monoclonal antibody, was radiolabeled with iodine-131 (131I) and technetium-99m (99mTc) to assess its biodistribution and internalization in Tn-expressing (Tn+) and wild-type (Tn-) LL/2 lung cancer cells. Selective accumulation and gradual internalization of ChiTn were observed in Tn + cells. Biodistribution in mice with both Tn + or Tn- lung tumors indicated that the uptake of radiolabeled ChiTn within tumors increased over time. Dual-labeling experiments with 99mTc and 131I showed different biodistribution patterns, with 99mTc exhibiting higher values in the liver, spleen, and kidneys, while 131I showed higher uptake in the thyroid and stomach. However, tumor uptake did not significantly differ between Tn + and Tn- tumors. To improve tumor targeting, Losartan, an antihypertensive drug known to enhance tumor perfusion and drug delivery, was investigated. Biodistribution studies in Losartan-treated mice revealed significantly higher radiolabeled ChiTn uptake in Tn + tumors. No significant changes were observed in the uptake of the control molecule IgG-HYNIC-99mTc. These findings demonstrate the enhanced tumor targeting of radiolabeled ChiTn in Losartan-treated mice with Tn-expressing lung tumors. They highlight the potential of ChiTn as a theranostic agent for cancer treatment and emphasize the importance of Losartan as an adjunctive treatment to improve tumor perfusion and drug delivery.
Article
Enhanced Tumor Targeting of Radiolabeled Mouse/Human Chimeric Anti-Tn Antibody in Losartan-Treated Mice Bearing Tn-Expressing Lung Tumors
https://doi.org/10.21203/rs.3.rs-3139336/v1
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published 12 Jan, 2024
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Monoclonal antibodies (mAbs) have been widely used in clinical practice for the diagnosis and treatment of various human pathologies, including cancer and infectious diseases, as well as for the modulation of the immune response (Buss et al., 2012; Hafeez et al., 2018; Kaplon & Reichert, 2021). The development of therapeutic mAbs in cancer has been facilitated by the identification of cell surface structures. These structures, which are either present or overly expressed in abnormal cells compared to their normal counterparts, act as targets for the effective function of mAbs (Martineau et al., 2018). Aberrant glycosylation is a common change in cancer cells, which causes incomplete elongation of the carbohydrate chains and unmasking of tumor-associated carbohydrate antigens on the cell surface (Hakomori, 2002). The Tn-antigen (CD175) is formed by an N-acetyl galactosamine (GalNAc) residue O-linked to the amino acids serine or threonine, present in the amino acid sequence of different glycoproteins, especially those of the mucin-type (Ju et al., 2011). This antigen is a cryptic determinant in cancer with high expressions in various epithelial cancers (ovarian, breast, prostate, colorectal, and lung cancers) (Hubert et al., 2011; Sedlik et al., 2016). Tn is associated with metastasis and cancer aggressiveness (Fu et al., 2016; Liu et al., 2019). Indeed, in Lewis lung cancer (LL/2), Tn antigen promotes tumor growth, immune evasion, and angiogenesis, resulting in larger and highly vascularized tumors (da Costa et al., 2021). This may be explained by the ability of LL/2 Tn + cells and LL/2 Tn + tumors to produce higher amounts of vascular endothelial growth factor (VEGF) compared to LL/2 wild type (Tn-) (da Costa et al., 2021). VEGF is considered to be the primary angiogenesis factor and has been shown to play a role in inducing extracellular matrix (ECM) synthesis and the angio-fibrotic switch in fibrosis (Larsson-Callerfelt et al., 2017; Kuiper et al., 2008; Zhang et al., 2019). Fibrosis, characterized by the excessive deposition of collagen, hyaluronan, and other ECM components, plays a critical role in tumor progression (Zhang et al., 2019). The fibrotic ECM has been identified as a significant contributor to the elevated solid stress observed in tumors (Jain et al., 2014). Unlike fluid pressure, this physical force is specifically contained within and transmitted by the solid phase of the tumor, resulting in the compression of blood vessels. This compression, coupled with the structural abnormalities of tumor blood vessels, renders them highly susceptible to collapse under the influence of the increased compressive forces. Consequently, the overall blood flow throughout the tumor mass is compromised (Liu et al., 2012; Chauhan et al., 2013; Jain et al., 2014; Nia et al., 2016). This reduction in perfusion has two major consequences: (i) compromised efficacy of treatment due to reduced drug delivery to tumors (Stylianopoulos et al., 2018), and (ii) increased tumor hypoxia, which promotes aggressive phenotypes, immunosuppression, and resistance to chemotherapy, radiation, and immunotherapy, all of which require oxygen to be effective (Jain, 2014). Therefore, strategies aimed at reducing solid stress and improving tumor perfusion are being explored as a means to improve the delivery and efficacy of cancer therapeutics. One such strategy is the use of Losartan, an FDA-approved antihypertensive agent that has been shown to improve tumor perfusion and drug delivery in mouse models of breast and pancreatic cancer by reducing the collagen and hyaluronan content within tumors (Diop-Frimpong et al., 2011; Chauhan et al., 2013; Zhao et al., 2019).
Several mAbs targeting the Tn-antigen with different specificities have been developed, some of which have demonstrated increased survival in mice inoculated with Jurkat (Ando et al., 2018), mammary (Hubert et al., 2011), and lung carcinoma cell lines (Wellinder et al., 2011). In addition to their inherent ability to inhibit tumor growth by inducing or stimulating the immune system (Scott et al., 2012; Vacchelli et al., 2013), the antitumor efficacy of mAbs can be significantly enhanced by conjugation with drugs (Chau et al., 2019), oligonucleotides (Dovgan et al., 2019), or radionuclides (Steiner & Neri, 2011; Audicio et al., 2011; Camacho et al., 2017; Perroni et al., 2021). A mouse/human IgG1 chimeric anti-Tn mAb, ChiTn, has been developed and used as an antibody-drug conjugate. Results showed that ChiTn internalizes in Tn + tumor cells in vitro and selectively accumulates in the tumor in vivo (Sedlik et al. 2016).
Imaging and biodistribution studies utilizing radioactive tracers have emerged as a highly sensitive and versatile approach, making it one of the main approaches for in vitro and in vivo studies of molecular-level processes. It allows analysis of pharmacokinetics, heterogeneity, antigen expression and engagement, and uptake of radiolabeled mAbs in tumors and other organs (Harsini & Rezaei, 2020; Perroni et al. 2021; Lin et al., 2021). Among commonly used radionuclides in nuclear medicine, iodine-131 (131I) stands out as a dual β and γ emitter with an 8.02-day half-life, being suitable for both tracer studies and therapeutic applications (Zhang et al., 2022). Additionally, technetium-99m (99mTc), with its 6-hour half-life and γ-ray emission, provides valuable capabilities for imaging and biodistribution studies (Duatti, 2021).
In this study, we aimed to explore the potential of a radiolabeled mouse/human IgG1 chimeric anti-Tn mAb (ChiTn) as a theranostic agent for cancer. We radiolabeled ChiTn with 131I and 99mTc, and evaluated its biodistribution and internalization in Tn + and Tn- (wild-type) cells. Furthermore, we investigated the potential use of Losartan as an adjunctive treatment to enhance the delivery and targeting of radiolabeled ChiTn to Tn + tumors in murine models of Tn + and Tn- LL/2 lung cancer.
ChiTn and IgG linker formation
The mouse/human IgG1 chimeric anti-Tn mAb (Chi-Tn) (Osinaga et al., 2000) was obtained from Laboratorio de Glicobiología e Inmunología tumoral / Institut Pasteur Montevideo. A human IgG (Sigma-Aldrich, USA) was used as a control molecule, which has no declared antigen-specificity. The conjugation reaction was initiated by adding a N-succinimidyl 6-(trifluoroacetyl) hydrazinopyridine-3-carboxylic acid hydrazino-protected form of succinimidyl ester (NHS-HYNIC-Tfa), synthesized in our laboratory (Garcia et al., 2014), and 100 nmol in 7 µL dimethylsulfoxide ((DMSO), J. T. Baker) were added to purified ChiTn or IgG control (10 nmol) in, 0.15 M pH 8 NaC2H3O2 buffer and incubated in darkness for 30 min at room temperature (RT). The ChiTn-HYNIC and IgG-HYNIC were subsequently purified by size-exclusion chromatography using NaCl 0.9% and a Sephadex G-25 (SEC) column (PD-10, Amersham Biosciences).
ChiTn and IgG radiolabeling
Radiolabeling of ChiTn-HYNIC or IgG-HYNIC with 99mTc was performed by adding 33.5 mol of tricine, 33 mol of SnCl2.2H2O, and 5 nmol of the antibody conjugate. Immediately, a Na99mTcO4− solution was added, and the mixture was incubated at RT for 30 min. Radiolabeling of ChiTn or IgG with 131I was achieved using the Pierce pre-coated iodination tubes (PPCIT) (Thermo Fisher Scientific, Waltham, MA, USA), and the “Example Protocol I: The Chizzonite Indirect Method for Iodination” protocol according to Thermo Fisher Scientific manual 0016379. Briefly, the PPCIT was wetted with 1 ml of Tris iodination buffer and decanted. Then, 10 MBq of 131I were added to the PPCIT and activated for 6 min at RT. Subsequently, 50 µg of ChiTn or IgG were added and the reaction mixture was incubated for 10 min at RT.
ChiTn and IgG radiolabeling controls, purification, and stability
Both radiolabeling were evaluated for radiochemical purity by 1) SEC equilibrated and eluted with normal saline, at 1.0 mL/min flow rate, and 2) High-performance liquid chromatography (HPLC) with a Protein-Pak 300 SW 7.5 mm x 30 cm column (Waters, USA) using isocratic mode with 0.05 M pH 7.0 phosphate buffer, 1 mL/min flow rate, equipped with UV and NaI(Tl) absorbance and scintillation detectors respectively. Technetium-99m radiolabeling was assessed using Instant thin-layer chromatography (ITLC) chromatography with various systems: ITLC-SG (Pall Corporation) combined with 0.9% NaCl, ITLC-SG saturated with bovine serum albumin (BSA, Sigma-Aldrich) and EtOH:NH3:H2O (2:1:5), as well as Whatman 1 paper (Whatman International Ltd) with methyl ethyl ketone (MEK). To ensure the integrity of the radiolabeled antibodies, the same HPLC system was employed. The radiolabeled antibodies were incubated in serum at 37°C for different time intervals, followed by HPLC analysis to evaluate their stability.
Cell culture
Wild-type (Tn-) murine lung cancer cell line LL/2 (ATCC), and LL/2 cells expressing the Tn antigen (Tn+) (da Costa et al., 2021), were grown in RPMI-1640 medium (Capricorn Scientific) supplemented with 10% Fetal Bovine Serum (FBS, Capricorn Scientific), 2 mM L-Glutamine, 100 U/mL penicillin and 100 µg/mL Streptomycin (Capricorn Scientific). All cultures were incubated at 37 ºC with 5% CO2 and a 95% humidity atmosphere.
Membrane-bound and cell internalization
Membrane-bound and cell internalization assay was performed by seeding 0.5 x 106 Tn- or Tn + cells with culture medium in 24 well plates and allowed to grow overnight. After washing once with culture medium, cells were incubated with ∼4 nM of ChiTn and/or IgG radioconjugated with 99mTc and/or 131I for 1, 2, 3, 4 (quadruplicates) and 24 h (octuplicate) at 37 ºC. Then, the reaction medium was aspirated and cells were washed twice with 500 µL cold Phosphate-buffered saline (PBS). Cells were washed with cold acid buffer solution (0.2 M pH 2 glycine buffer) for 5 min to remove membrane-bound radioconjugated antibodies. The remaining internalized radioconjugated antibodies were obtained by lysis with 1 M NaOH for 5 min. Membrane-bound and internalized radioactivities were measured, and the results were analyzed, according to Radioactivity measurements and Statistical analysis sections.
Animals and tumor induction
C57BL/6 female mice were purchased from URBE Laboratories (Uruguay). Mouse handling, care, and experiments were in accordance with ethical principles adopted by National Committee on Animal Research (CNEA, Uruguay) (protocol approved by the Commission of Ethics in the Use of Animals, Facultad de Ciencias (CEUA) number 240011-001327-18). Tn- or Tn + LL/2 cells (2 × 105/100 µL) were inoculated on the flank region of each mouse via subcutaneous (s.c.) or intradermal (i.d.) injection (5 mice/group). The animals were followed daily for at least 1 month, evaluating tumor growth. All procedures were confirmed to comply with ARRIVE guidelines.
Losartan treatment
Losartan pills (50 mg/pill) (Losartan potassium) were ground using a mortar and pestle, and dissolved in PBS to obtain a concentration of 2.5 mg/mL. The solutions were then filtered and stored in a sterile container. Animals were treated daily with an intraperitoneal (i.p.) injection of Losartan (20 mg/kg) or saline (control) for 9 days (Diop-Frimpong et al., 2011; Chauhan et al., 2013; Zhao et al., 2019).
Biodistribution studies
When tumors reached 500–1000 cm3 in size (15–20 days), mice from the same cohort received 0.74-3 MBq of ChiTn-HYNIC-99mTc or IgG-HYNIC-99mTc (1–4 µg, in 100 µL PBS), and/or approximately 185 kBq of ChiTn-131I or IgG-131I (1 µg, in 100 µL PBS) via the tail vein. After 4, 24, and 48 h, the animals were euthanized with anesthetic drugs (xylazine-100 mg/Kg and ketamin-300 mg/Kg). Blood, urine, and feces were collected, tumor and selected organs were excised, rinsed of residual blood, weighed and their radioactivity was measured according to the Radioactivity measurements section. Results were expressed as percent injected dose (% ID) and per gram of tissue (% ID/g).
Radioactivity measurements
Gamma-ray measurements were acquired using a high-purity germanium detector (HPGe) (Canberra) and appropriate low-background shielding. The HPGe has the following features: 20% counting efficiency (at 1332 keV) and FWHM resolution < 0.85 keV (at 122 keV), enabling simultaneous in vitro and/or biodistribution measurements of multiple gamma-emitting radionuclide (Knight et al., 2019; Tassano et al., 2021). Results are reported for 99mTc (140.5 keV, 89%) lower limit of detection (LLD) 110 Bq, and 131I (364.5 keV, 81.7%) LLD 1.0 kBq for 30-second measurements.
Statistical analysis
To evaluate the statistical significance of differences in medians, we used the Mann-Whitney U test (MW) with Bonferroni correction. The following annotation was used for statistical significance, ns: p < = 1e+ 00; *: 1e− 02 < p < = 5e− 02; **: 1e − 03 < p < = 1e− 02; ***: 1e− 04 < p < = 1e− 03; ****: p < = 1e− 04, also, a 95% confidence interval (CI) was applied. We performed all statistical analyses and data visualizations using multiple packages within Spyder v. 4.1.5 (Raybaut, 2009), an integrated development environment (IDE) for scientific programming in Python v. 3.8.5 (Python Software Foundation). The packages utilized include Pandas (McKinney, W., 2010) for data manipulation, Matplotlib (Hunter, J. D., 2007) for data visualization, NumPy (Harris et al., 2020) for numerical computations, Seaborn (Waskom et al., 2021) for advanced data visualization, and Statannotations (Charlier et al., 2022) for statistical annotations.
Radiolabeling and in vitro quality controls
Both 131I and 99mTc radiolabeled ChiTn or IgG antibodies were purified and their chemical identity was determined by SEC, HPLC, and ITLC. SEC analysis showed a retention volume of the radiolabeled antibodies between 3–4 mL, while free 99mTc and free 131I showed a retention volume between 8–9 mL. HPLC profile showed retention times of 8.5, 8.9, and 9.0 min for ChiTn-HYNIC-99mTc, ChiTn-131I, and unlabeled ChiTn respectively (Fig. 1). Radiochemical purity exceeded 92% in all the experiments. The analysis conducted by ITLC demonstrated that within the residual 8%, nearly two-thirds were attributed to 99mTcO2.H2O, while the remaining one-third was associated with 99mTc-tricine. The radiochemical stability exhibited a gradual decrease over time, with an average of 89% (SD 2.1) and 90% (SD 2.08) of antibodies retaining their radiolabeling with 99mTc and 131I, respectively, after a 48 h incubation period in serum at 37 ºC (Fig. 1, lower right). Similar results of radiochemical purity and stability were obtained for IgG-HYNIC-99mTc (data not shown).
Membrane-bound and cell internalization
Results for the membrane-bound, internalization, and uptake ratio between Tn + and Tn- LL/2 cells for radioconjugated ChiTn and/or IgG are shown in Fig. 2, and data are available at https://zenodo.org/record/8110446. A gradual, time-dependent increase in both membrane-bound and intracellular activities was observed for ChiTn-HYNIC-99mTc during the 24 h experiment at 37°C (Fig. 2A). This increase was found to be significantly higher in Tn + cells compared to Tn- cells (p < = 1e-04 at 24 h). Similarly, when IgG-HYNIC-99mTc and ChiTn-131I were co-incubated, a similar trend of gradual increase was observed for ChiTn-131I, with significantly higher activities observed in Tn + cells (1e-03 < p < = 1e-02 at 24 h). However, no specific trend or significant differences were observed between Tn + and Tn- cells when using IgG-HYNIC-99mTc (Fig. 2B). The total cell-associated uptake of ChiTn-HYNIC-99mTc was approximately 100% higher in Tn + cells compared to Tn- cells, while for ChiTn-131I, the difference was approximately 50% (Fig. 2C at 24 h). In contrast, no significant difference favoring Tn + cells was observed for IgG-HYNIC-99mTc (Fig. 2C).
Biodistribution studies
The results of the biodistribution of ChiTn-HYNIC-99mTc in female C57BL/6 inoculated s.c. or i.d. with Tn- or Tn + LL/2 cells obtained at 4, 24, 48 h post-injection, are shown in Fig. 3 and Sup. Figure 1, and data are available at https://zenodo.org/record/8110446. An increase in Tn + tumor uptake occurred between 4 h (4.4% ± 1.6 ID/g) and 24 h (10.5% ± 2.3 ID/g) post-injection, remainining stable at 48 h (11.3% ± 2.5 ID/g). No significant differences in the uptake were found between Tn- and Tn + or between s.c. or i.d. tumors. Furthermore, there were no significant differences between Tn- and Tn + or between s.c. or i.d. tumors concerning the ratios of tumor-to-blood (T/B) and tumor-to-muscle (T/M). The remaining organs showed a typical antibody biodistribution profile, with liver uptake and hepatobiliary elimination (Sup. Figure 1).
The biodistribution of double-labeled ChiTn with 99mTc and 131I at 48h post-injection (Fig. 4 above) resulted in a profile with higher values for 99mTc in the liver, spleen, kidneys and a tendency to show higher values in Tn- and Tn + tumors, although this was not significant. The 131I shows a high preference for the thyroid and is also significantly higher in the stomach. No significant differences were found in the T/B and T/M ratios. Also, the biodistribution of the co-injection of IgG-HYNIC-99mTc and ChiTn-131I was performed at 48 h (Fig. 4 below). Most of the organs showed a higher %ID/g of IgG-HYNIC-99mTc compared to ChiTn-131I, even in the Tn- tumor, but not in the Tn+. Increased uptake was also found in the thyroid by ChiTn-131I. No significant differences were found in the T/B and T/M ratios, for both biodistributions. All values, except for thyroid, are comparable to Fig. 3 and Sup. Figure 1. Data available at https://zenodo.org/record/8110446.
Finally, the biodistribution of ChiTn-HYNIC-99mTc and IgG-HYNIC-99mTc at 48 h in mice pre-treated with Losartan is shown (Fig. 5), data available at https://zenodo.org/record/8110446. Although the profile was similar to Fig. 3 and 4, interestingly a higher uptake was observed in Tn+ than in Tn- tumors for ChiTn-HYNIC-99mTc. Also, higher T/B and T/M ratios were observed between Tn+ and Tn- tumors for ChiTn-HYNIC-99mTc. No significant differences between Tn+ and Tn- tumors were observed for IgG-HYNIC-99mTc. The average results and their standard deviation are summarized in Table 1.
Table 1. Total 48 h biodistribution results for Tn+ and Tn- tumors, including %ID/g averages, standard deviations, tumor-to-blood (T/B), and tumor-to-muscle (T/M) ratios.
Effective drug delivery to tumors is often hindered by several barriers, such as physical barriers, low oxygen levels, and acidic pH, impeding the penetration of anticancer agents (Curti, 1993; Sriraman et al., 2014). Physical barriers include the interstitial transport of antibodies in normal and neoplastic tissues (Curti, 1993, Libuti et al., 2018). Such barriers could explain why some drugs, effective in vitro, fail to work in vivo (Jain, 1994; Anchordoquy et al., 2017). To address this, we investigated the potential of Losartan to enhance the delivery of the ChiTn antibody to Tn + tumors by reducing solid stress (Diop-Frimpong et al., 2011, Chauhan et al., 2013, Zhao et al., 2019). We evaluated the radiolabeled mAb ChiTn intending to use it as a tracer, but also as a potential cancer theranostic agent.
Our experimental findings demonstrate that the radiochemical purity of both 99mTc and 131I radiolabeled antibodies remained consistently above 92% even after a 48 h incubation period in serum at 37 ºC (Fig. 1). These results align with previous studies that reported similar levels of radiochemical purity and stability over time using the same antibody labeling methodology with 99mTc (Camacho et al., 2017; Perroni et al., 2021) and 131I (Vinod et al., 2021).
Different binding and internalization capacities of the ChiTn mAb to tumor cell lines expressing the Tn-antigen have been observed (Hubert et al., 2011; Sedlik et al., 2016’, Castro et al., 2021). Additionally, it is rapidly internalized by Tn + tumor cells and primarily localizes in early endosomes (Hubert et al., 2011; Sedlik et al., 2016). Our results show that membrane-bound and internalization of the labeled antibody in Tn + and Tn- LL/2 cells exhibit slower kinetics than the unlabeled antibody, but are similar to those of other radiolabeled antibodies (Kuo et al., 2018; Camacho et al., 2014). The 99mTc labeling process involves the reaction of HYNIC with, for example, the ε-amino group of antibody lysines (Meszaros et al., 2010; Garcia et al., 2016). Similarly, for the chemical oxidation process used in 131I labeling, sodium iodide is converted into a reactive form that can be incorporated into the tyrosyl groups of the antibody (Gupta et al., 2014; Feng et al., 2022). Therefore, modification by radioactive labeling can alter the physical and chemical properties of the antibody, which can affect its interaction with cells (Tolmachev et al., 2014).
ChiTn-HYNIC-99mTc showed persistent membrane binding after 24 h, favoring Tn + cells significantly (Fig. 2A). Notable Tn + internalization was observed after 4 h (Fig. 2A). ChiTn-131I yielded similar results unlike the control IgG-HYNIC-99mTc, which showed minimal binding and internalization with no significant Tn + and Tn- differences (Fig. 2B). Figure 2C illustrates the disparities in Tn+/Tn- ratios.
Although intermediate affinities of radiolabeled antibodies may hinder binding and internalization, studies by Rudnick et al. have shown that low-affinity mAbs can penetrate solid tumors more efficiently (Rudnick et al., 2011). To investigate the uptake and retention ability of radiolabeled ChiTn and IgG in Tn + and Tn- tumors in vivo, we conducted various biodistribution studies.
The biodistribution results of ChiTn-HYNIC-99mTc indicated a typical distribution pattern for 99mTc radiolabeled antibodies through HYNIC, as reported by Camacho et al. (2014). The radiotracer exhibited slow clearance in the bloodstream, liver, heart, and lungs, and sustained tumor uptake up to 48 h (11.3 ± 2.5%ID/g), with no significant differences between Tn + and Tn- tumors or T/B and T/M ratios (Fig. 2, 3, and Sup. Figure 1). Similar results were seen for i.d. and s.c. injections of LL/2 Tn + and Tn- cells in mice.
To assess the impact of the radioactive labeling method on biodistribution (Williams, 2014), we performed dual-labeling of the ChiTn antibody with 99mTc and 131I. This enabled simultaneous measurement of biodistribution using gamma spectrometry (Tassano et al., 2021), thereby minimizing inter-mouse, inter-measurement, and handling variability (Knight et al. 2019). Double-labeled ChiTn exhibited higher values for 99mTc in the liver, spleen, and kidneys, with a slight tendency for higher values in both Tn- and Tn + tumors (Fig. 4 above), but this was not significant. The 131I exhibited a high affinity for the thyroid and was also significantly higher in the stomach. The T/B and T/M ratios did not show any significant differences.
When radioiodinated antibodies are taken up by cells, they are quickly broken down in lysosomes, resulting in the release of monoiodotyrosine into the extracellular space. This metabolite is further broken down by deiodination enzymes, which release free radioiodide into the bloodstream (Press et al., 1996). The radioiodide is then taken up by any tissues expressing the sodium-iodide (Na+/I−) symporter, which is present in the thyroid gland and stomach. The lysosomal degradation of radioiodinated antibodies leads to the rapid clearance of radioiodine from all tissues, except those that metabolize or process iodine (Vivier et al., 2018). This occurrence leads to diminished activity concentrations within the tumor tissue in comparison to residualizing radiolabels that employ the radiometal 99mTc (Deyev et al., 2020).
The accumulation of the antibody within tumors ideally would solely rely on the specific target antigen. However, challenges arise when non-specific factors contribute to overall tumor uptake. One example is the enhanced permeability and retention (EPR) effect, which occurs due to rapid and irregular angiogenesis, resulting in antibodies passively extravasating into the tumor tissue through the leaky vasculature (Heneweer et al., 2011; Fang et al., 2011). These non-specific contributions to tumor uptake can vary significantly between tumor models, within a single tumor (intra-tumoral heterogeneity), or as a result of different responses to treatment (inter-tumoral heterogeneity). Consequently, the sensitivity of these techniques may be reduced, increasing the likelihood of false discoveries (Börjesson et al., 2006).
We conducted an additional experiment to assess non-specific uptake by comparing the biodistribution of Tn-specific ChiTn-131I with that of non-specific IgG-HYNIC-99mTc. Most of the organs showed a higher % ID/g of IgG-HYNIC-99mTc compared to ChiTn-131I (Fig. 4 below). This is expected since the radioactive label with 131I tends to be more unstable in vivo, leading to increased uptake in the thyroid and stomach, as well as greater clearance from organs, as previously discussed. Nevertheless, the Tn + tumor demonstrated a comparable uptake of ChiTn-131I to that of IgG-HYNIC-99mTc, while a reduced uptake was observed in Tn- tumors. Once again, no distinct advantage in uptake was noted for the Tn-specific ChiTn as compared to the non-specific IgG. This observation is further corroborated by the similarities in the tumor-to-blood (T/B) and tumor-to-muscle (T/M) ratios (Fig. 4 below).
The presence of solid stress may be a contributing factor hindering the efficient delivery of antibodies to tumors, particularly in highly vascularized tumors such as LL/2 Tn+. These tumors produce higher levels of VEGF compared to LL/2 wild type (Tn-), as demonstrated by da Costa et al. (2021). VEGF is recognized as a key factor in angiogenesis, capable of inducing ECM synthesis and promoting the angio-fibrotic switch in fibrosis, as documented by Larsson-Callerfelt et al. (2017), Kuiper et al. (2008), and Zhang et al. (2019). The fibrotic ECM significantly contributes to elevated solid stress within tumors (Jain et al., 2014).
To assess this phenomenon, biodistribution studies were conducted after pretreatment with Losartan, a compound known to alleviate solid stress in fibrotic tumors (Diop-Frimpong et al., 2011; Chauhan et al., 2013). Previous research has indicated that Losartan treatment does not alter VEGF levels or microvessel density. However, it has been shown to significantly enhance the percentage of perfused blood vessels (Zhao et al., 2019).
The % ID/g values indicate that the biodistribution profiles of normal tissues in the Losartan pre-treated mice (Fig. 5) were comparable to those of the untreated counterparts at 48 h post-injection for both IgG-HYNIC-99mTc and ChiTn-HYNIC-99mTc, which aligns with the findings of Chauhan et al. (2013) where Losartan treatment does not affect accumulation in normal tissues. However, a slightly higher uptake in the kidneys and elimination through urine and feces were observed for ChiTn-HYNIC-99mTc (Fig. 5). Additionally, ChiTn-HYNIC-99mTc demonstrated a higher blood clearance compared to IgG-HYNIC-99mTc, as evident in Fig. 5, consistent with the outcomes of the previous biodistribution studies where ChiTn-HYNIC-99mTc values ranged from 15 to 20% ID/g and IgG-HYNIC-99mTc values ranged from 22 to 30% ID/g at 48 h in bloodstream (Fig. 3, 4, and 5).
In mice pre-treated with Losartan, a significantly higher uptake of ChiTn-HYNIC-99mTc was observed in Tn + tumors (%ID/g 14.9 ± 2.1) compared to Tn- tumors (%ID/g 7.9 ± 1.2) (p ≤ 0.05), as indicated in Table 1 and Fig. 5. However, there was no significant change in the uptake of IgG-HYNIC-99mTc in either tumor type. These findings are further supported by the T/B and T/M ratios (Table 1 and Fig. 5). ChiTn-HYNIC-99mTc demonstrated significantly higher ratios in favor of Tn + over Tn- tumors (p ≤ 0.01 and p ≤ 0.05, respectively), while IgG-HYNIC-99mTc did not show the same pattern. Specifically, the T/B ratio in Tn + tumors increased from 0.8 ± 0.5 in untreated mice to 1.2 ± 0.2 in mice pre-treated with Losartan for ChiTn-HYNIC-99mTc. The T/M ratio also exhibited a moderate increase from 7.7 ± 3.3 to 10.4 ± 3.3.
These values indicate a substantial increase in Tn + uptake compared to Tn- in mice pre-treated with Losartan, with an average of 88% higher uptake in Tn + tumors. Additionally, there was a significant enhancement in the T/B ratio, with a 50% increase. Similarly, the T/M ratio exhibited a 35% increase in Tn + tumors. All of these values closely align with the findings reported by Chauhan et al. (2013), where Losartan treatment resulted in a 74% increase in the accumulation of the small-molecule chemotherapeutic 5-FU in AK4.4 pancreatic tumors.
The findings indicate that LL/2 Tn + tumors exhibit elevated solid stress compared to their wild-type (Tn-) counterparts. As a result, Losartan is expected to be more effective in alleviating this stress specifically in Tn + tumors. Additionally, the radiolabeled ChiTn mAb demonstrates enhanced penetration into this tumor type, allowing for increased interaction with the Tn antigen. In contrast, nonspecific IgG lacks the capability to undergo specific binding and subsequent internalization into tumor cells, leading to a lack of this effect. Its uptake primarily occurs through nonspecific binding or due to its higher concentration in the bloodstream.
In conclusion, our study investigated the use of radiolabeled mouse/human chimeric anti-Tn antibody (ChiTn) as a tracer and cancer theranostic agent in combination with Losartan in mice with Tn-expressing lung tumors. We found that radiolabeled ChiTn exhibited greater binding and internalization in Tn + tumor cells compared to Tn- cells. Furthermore, Losartan treatment improved the in vivo uptake of radiolabeled ChiTn in Tn + tumors, suggesting enhanced tumor targeting. However, there were no significant differences in biodistribution between Tn + and Tn- tumors for radiolabeled IgG unspecific control molecule. The study provides insights into the potential use of radiolabeled ChiTn in combination with Losartan for improved delivery and targeting of Tn + tumors, which may have clinical implications for the treatment of epithelial cancers expressing the Tn-antigen. Further studies are warranted to explore the theranostic efficacy of this approach and optimize the radiolabeling and targeting strategies.
Acknowledgements
This work is part of the Master's and Doctoral Program in Medical Sciences (Proinbio) from the Facultad de Medicina at the Universidad de la Republica, Uruguay, where MSc. Marcos Tassano is pursuing his doctorate in medical science. We would like to extend special thanks to MD-PhD Roger Chammas, Professor of Oncology and Deputy Dean at the Faculty of Medicine of the University of São Paulo and the Cancer Institute of the State of São Paulo, for his insightful advice on the use of Losartan for tumor delivery.
Author contributions
M.T., X.C., C.P., M.C., M.F.G., M. F., J.P.G. and P.C. participated in the antibody radiolabeling and in vitro/in vivo studies in both models. T.F., V.D.C., and E.O., developed cell lines, tumor models and the ChiTn antibody. M.T. drafted the manuscript. All authors participated in the editing and review of the manuscript.
Data availability statement
Comprehensive datasets for in vitro and biodistribution assays can be accessed via this link: https://zenodo.org/record/8110446.
Additional Information
The authors declare no competing interests.
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No competing interests reported.