A Tetramethylpyrazine Releasing Hydrogel can Potentiate CAR-T Cell Therapy against Triple negative breast cancer by Reprogramming Tumor Vasculatures

doi:10.21203/rs.3.rs-2223773/v1

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Research Article

A Tetramethylpyrazine Releasing Hydrogel can Potentiate CAR-T Cell Therapy against Triple negative breast cancer by Reprogramming Tumor Vasculatures

https://doi.org/10.21203/rs.3.rs-2223773/v1

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Background: Irregular vasculature of solid tumors has proven to be a pivotal factor restricting their response to chimeric antigen receptor-T (CAR-T) cell therapy because it is tightly associated with hypoxia and other biological barriers.

Methods: Herein, an injectable hydrogel composed of poly (ethylene glycol) dimethacrylate (PEGDMA) and ferrous chloride (FeCl₂) responding to endogenous hydrogen peroxides (H₂O₂) is developed to enable sustained intratumoral release of Chinese herbal extracts tetramethylpyrazine (TMP). then the effect and mechanism of angiogenesis and tumor vasculature maturation by TMP@PEGgel were evaluated by immunofluorescence, flow cytometry, western blotting, real-time PCR and enzyme linked immunosorbent assay. Furthermore, tumor infiltration, survival, and effector function of CAR-T cell were investigated by in vitro different cell assay and in vivo tumor xenografts mouse models.

Results: Upon being fixed inside tumors with the PEGDMA based hydrogel, TMP can remodel tumor vasculature by simultaneously promoting angiogenesis and dilating tumor vasculature via activating vascular endothelial growth factor (VEGF) expression and the endothelial nitric oxide synthase/nitric oxide (eNOS/NO) axis inside vascular endothelial cells and thus attenuate tumor hypoxia in two murine xenografts bearing human triple negative breast cancer (TNBC). Resultantly, treatment with TMP fixation potentiate the tumor suppression effect of intravenously injected epidermal growth factor receptor expressing CAR-T (HER-1-CAR-T) cells toward two TNBC tumor xenografts by promoting their tumor infiltration, survival, and effector function.

Conclusion: This study highlights that TME remodeling through reprogramming tumor vasculature with TMP@PEGgel treatment is a promising and robust strategy to reinforce the therapeutic potency of CAR-T cell therapies toward solid tumors.

chimeric antigen receptor-T cell therapy

injectable hydrogel

triple-negative breast cancer

tetramethylpyrazine

tumor vasculature reprogramming

Triple-negative breast cancer (TNBC) lacking of the expression of progesterone receptor, estrogen receptor and human epidermal growth factor receptor 2 (HER2) is the most aggressive subtype of breast cancers [1, 2]. It has been reported that 45-70% of TNBC patients exhibit epidermal growth factor receptor (EGFR, also known as HER1) overexpression, which has been utilized as the target for inventing new therapeutics to expand our arsenal against TNBC [3]. Besides of developing small molecule EGFR inhibitors (e.g., Erlotinib, Icotinib, Gefitinib etc.), engineering of HER1-CAR-T cells represents a promising treatment modality as its potent capacity to selectively eradicate HER1 positive TNBC cancer cells [4-7]. However, similar to other CAR-T cell therapies in treating corresponding solid tumors, the therapeutic efficacy of HER1-CAR-T cells remains far from satisfactory because the complex tumor microenvironment (TME) of solid TNBC tumors would not only physically restrict their intratumoral infiltration, but impair their expansion, survival and effector functions via distinct mechanisms [8-12]. Recent progresses have demonstrated that concurrent delivery of CAR-T cells together with stimulatory cytokines or TME remodeling formulations could effectively potentiate these CAR-T cells against corresponding solid tumors by removing tumor immunosuppression [13-17]. Therefore, development of effective TME remodeling strategies is of great practical promise to enhance the therapeutic potency of HER1-CAR-T cells [18].

In the past decades, intensive efforts have been devoted to find out efficient TME remodeling strategies in both clinical and preclinical explorations [19]. Normalization of tumor vasculature through different mechanisms has attracted tremendous research interests since the abnormal tumor vasculature is tightly associated with tumor hypoxia, tumor acidity, tumor immunosuppression and many others [20-22]. It is therefore shown to be versatile in directly suppressing tumor growth and benefiting other cancer treatment modalities by promoting the tumor accumulation and intratumoral diffusion of distinct cancer therapeutics (e.g., small molecule chemotherapeutics, nanotherapeutics). Recently, we uncovered that tumor targeted delivery of Erlotinib with a pH-responsive nanocarrier could effectively remodel tumor vasculatures through inhibiting HER1/extracellular regulated protein kinases (ERK)/AKT serine/threonine kinase 1 (AKT) axis, and thus attenuate tumor hypoxia for enhanced photodynamic treatment of TNBC tumors [23]. However, till now, there is no study on exploring the potency of tumor vasculature reprogramming in potentiating CAR-T cell therapy and other adoptive cell transferring therapies [24].

In addition, increased intratumoral levels of nitrogen oxide (NO) and vascular endothelial growth factor (VEGF) through distinct mechanisms could effectively remodel tumor vasculature due to their potency in dilating blood vessels and in regulating angiogenesis, respectively [19, 25-27]. Tetramethylpyrazine (TMP), an alkylpyrazine isolated from traditional Chinese medicine, Ligusticum wallichii (Chuan Xiong), has been used for the treatment of varying vascular diseases in China for ~40 years, probably ascribing to its potency in promoting VEGF expression and NO production in vascular endothelial cells [28]. Therefore, in this study, water soluble TMP molecules were in situ fixed with endogenous hydrogen peroxide (H₂O₂) responsive hydrogels composed of PEGDMA and FeCl₂ for long-term tumor vasculature remolding. It is found that obtained TMP loaded PEGDMA hydrogel (coined as TMP@PEGgel) upon intratumoral fixation could effectively remodel tumor vasculature through activating the VEGF expression and the eNOS/NO axis of vascular endothelial cells and thus enable sustained tumor hypoxia relief. Furthermore, such TMP@PEGgel treatment was shown to be capable of enhancing the infiltration of intravenously infused HER1-CAR-T cells and thus promoting their survival and effector functions inside MDA-MB-468 and MDA-MB-231 TNBC tumors inoculated on Balb/c nude mice. It was further shown that our TMP@PEGgel assisted HER1-CAR-T cell therapy could synergically suppress the growth of these two TNBC tumors. This study therefore highlights that remodeling of tumor vasculature with TMP treatment is a promising strategy to reinforce the therapeutic potency of HER1-CAR-T cell therapies toward TNBC tumors.

Materials and reagents

Poly(ethylene glycol) dimethacrylate (PEGDA), FeCl₂, rhodamine B were purchased from Sigma-Aldrich. Tetramethylpyrazine (TMP) was purchased from MCE medchemexpress. The LDH cytotoxicity assay kit and MTT assay kit was purchased from Beyotime Biotechnology. The ELISA kits were purchased from Dakewe. anti-CD3/CD28 beads was purchased from Invitrogen. Fetal bovine serum (FBS) and all the cell cultural medium were purchased from Thermo Fisher Scientific Inc. CCK-8 assay was purchased from Dojindo Laboratories. CAR-related plasmids were purchased from Icareab.

Preparation and characterization of PEGgel

PEGDMA (10 wt%) was mixed with FeCl₂ (1.6 mM) and H₂O₂ (0.5 mM) using a vortex mixer for 10s, then incubated at room temperature till the gel formation. The photographs were obtained at 1 min post the mixing. The microscopic structure of the as-prepared hydrogel was observed by the scanning electron microscopy (SEM, Zeiss Gemini 500). TMP release was evaluated at 37°C in PBS. The released TMP was analyzed with an ultraviolet-visible spectrophotometer (PerkinElmer).

Cell experiments

MDA-MB-468, MDA-MB-231, HUVEC and HAEC cells were obtained from ATCC, and maintained following the standard procedures.

T cells were obtained from PBMCs from healthy adult donors with signed agreements. In brief, PBMCs isolated from whole blood using ficoll density gradient centrifugation were stimulated in AIM V^TM complete medium supplemented with anti-CD3/CD28 beads (2.5μL/mL) with 5% CO₂ at 37 ℃. 2 week later, T cells were transduced with lentivirus which contained con-CAR or HER1-CAR. Afterwards, both CAR-T cells were collected after being stimulated with appropriate antibodies and antigens according to the standard methods [29]. Then, the CD3ζ expression levels on these obtained con-CAR-T and HER1-CAR-T cells were evaluated by using real-time PCR and western blotting assays to identify their successful construction by according to previous used method.

Then, the effects of hypoxia incubation on the cell viability of these two CAR-T cells were investigated by incubating these CAR-T cells seeded in 96-well plates (5×10⁴ cells per well) at both hypoxia condition (<2% O₂) and normoxic condition (21% O₂) for 24 h. The cell viability of these cells post corresponding treatments were determined by the standard MTT assay.

To evaluate the influence of hypoxia incubation on the specific cell killing of HER1-CAR-T cells, TNBC cells pre-seeded in the 96-well plate (1×10⁴ cells per well) for 12 h were co-cultured with HER-1CAR-T cells at varying feeding ratios under hypoxic condition or normoxic conditions for 24 h. Later, the concentration of LDH, released by dead TNBC cells, in the supernatant of each well was quantified by using commercial LDH cytotoxicity assay, while the secretion levels of IL-2, TNF-α and IFN-γ was determined by using corresponding ELISA kits.

To assess the cytotoxicity of TMP on endothelial cells, both HUVEC and HAEC pre-seeded in the 96-well plate (1×10⁴ cells per well) were incubated with fresh medium supplemented with different concentrations of TMP at 37 ^oC for another 24 h. Then, their cell viability was measured by the Cell Counting Kit-8 (CCK-8) method.

To assess the potency of TMP treatment in activating VEGF expression and eNOS/NO axis, both HUVEC and HAEC were seeded in the 6-well plates and incubated for 12 h before being incubated with f TMP (500 nM) at 37 ^oC for another 24 h. Then, the expression levels of VEGF, p-eNOS, eNOS, β-Actin in these cells was measured by western blotting by according to the manufacturers’ instruction. Another batch of cells with same treatments were used to evaluate the effect of TMP treatment on the measure NO production using commercial NO assay kit.

In vivo animal experiments

Female Balb/c nude mice (4-week-old) was purchased from Nanjing University’s model animal research center. Nanjing Normal University’s medical ethics committee approved the animal experiments in this study. TNBC cells (5×10⁶) were subcutaneously injected into the mammary fat pad of each mouse to generate TNBC cell-line-derived tumor model.

For in vivo sustained release study, Rhodamine B@PEGgel and free rhodamine B solution (rhodamine B = 1 mg/kg) were intratumorally injected into the TNBC tumors. At 15 min, 3 days, and 10 days post corresponding injection, tumors of treated mice were excised, cryosectioned, counterstained with DAPI being observed on a confocal microscopy for recording the rhodamine B fluorescence according to the standard method.

For in vivo tumor hypoxia evaluation related study, TNBC tumor bearing mice with intratumoral injection of TMP@PEGgel, PEGgel and TMP (TMP = 1 mg/kg) were sacrificed at 3 days, 10 days post corresponding injection for preparing tumor slices. Then, these tumors were stained with anti-HIF-1a and anti-CD31 primary antibodies and corresponding secondary antibodies by following the procedures provided by the manufacturers, followed by being observed using confocal microscopy.

For pimonidazole staining, another batch of mice with same treatments were intraperitoneally injected with commercial pimonidazole at 90 min before they were euthanized. Later, tumors were collected for hypoxia staining by using an hypoxyprobe-1 plus kit.

For evaluating the in vivo tumor infiltration, survival and effector functions of HER1-CAR-T cells, 30 mice bearing TNBC tumors were randomly divided into six groups and received following treatments: 1) PBS, 2) con-CAR-T injection, 3) HER1-CAR-T injection, 4) PEGgel fixation + HER1-CAR-T injection, 5) TMP injection + HER1-CAR-T injection, 6) TMP@PEGgel fixation + HER1-CAR-T injection. And then mice received i.v. injection of 5×10⁶ con-CAR-T cells, HER1-CAR-T cells, both with intrinsic expression of RFP at 1 day. At 3 day and 10 days post TMP@PEGgel injection, tumors were cryosectioned or homogenized to evaluate the tumor infiltration and survival of both CAR-T cells by recording the RFP fluorescence by using confocal microscopy and flow cytometry, respectively. In addition, tumors were wet-weighted and homogenized in lysis buffer for measuring the secretion of IL-2, TNF-α and IFN-γ by using corresponding ELISA kits according to manufacturers’ instruction.

For in vivo combined treatment, 6 groups TNBC tumor bearing mice (~100 mm³) received same intratumoral injections of TMP@PEGgel, PEGgel and TMP at day 0 as aforementioned, but received 3 times i.v. injection of con-CAR-T cells or HER1-CAR-T cells (5×10⁶ cells per injection) at 1, 2, 3 days. Digital caliper was used to record the length and width of each tumor every week for calculating tumor volumes by following the formula of Volume=1/2 × Length × Wideth× Wideth. Digital balance was used to monitor the body weight of each mouse every week.

Statistical Analysis

Statistical analysis performed by GraphPad Prism software. All results are presented as the mean ± standard error of mean, and statistic quantification analyzed by t-test.

Hypoxia inhibits the effector functions of HER1-CAR-T cells

To construct HER1-CAR-T cells, a third‐generation CAR expressing plasmid containing CD8 signal, CD8 Hinge, CD28, 4-1BB, and CD3ζ expressing sequences was first fused with the sequence encoding HER1 specific single-chain fragment variable (scFv) (Figure 1A-B). Then, the obtained HER1-CAR expressing plasmid was encapsulated with Lentivirus by using 293T/17 cells and then utilized to transduce T cells isolated and differentiated from healthy donor’s human peripheral blood mononuclear cells (PBMCs) according our previously used method [30]. Meanwhile, non-specific control CAR T (con-CAR-T) cells were obtained by transducing T cells with the plasmid encoding the non-specific third-generation CAR through the aforementioned method. The successful construction of these two CAR-T cells were confirmed by the emergence of CD3ζ signals at both transcription and protein levels detected by using real-time polymerase chain reaction (PCR) and western blot analysis, respectively (Figure 1C-D). The larger molecular weight of CD3ζ of HER1-CAR-T cells compared to that of con-CAR-T cells could further validate the successful fusion of CAR with anti-HER1 scFv.

The impacts of hypoxia incubation on the cell viabilities and effector functions of these newly engineered HER1-CAR-T cells were then carefully investigated. By using the commercial MTT assay kit, it was found that the cell viabilities of con-CAR-T and HER1-CAR-T cells after being incubated under the hypoxia condition (<2% O₂) for 24 h were only ~67% and ~72% to corresponding cells incubated under the normoxic condition (21% O₂) (Figure 1E). In addition, it was shown that co-incubation of HER1-CAR-T cells with HER1 overexpressing human MDA-MB-468 and MDA-MB-231 TNBC cells under the normoxic condition for 24 h would lead to severe TNBC cell death as indicated by the cytotoxicity assay. In contrast, it was shown that the hypoxia incubation condition remarkably diminished the specific cell killing capacity of HER1-CAR-T cells toward co-cultured MDA-MB-468 and MDA-MB-231 cells. Meanwhile, it was shown that con-CAR-T cells exhibited negligible disturbance on the cell viability of these co-cultured TNBC cells (Figure 1F-G). Furthermore, it was uncovered that HER1-CAR-T cells co-incubated with MDA-MB-468 and MDA-MB-231 cells at varying feeding ratios exhibited significant secretion of interleukin 2 (IL-2) (Figure 1H), which in turn is reported to be able to promote the expansion of T cells [31]. In addition, such treatment was also capable of promoting the secretion of tumor suppression cytokines of tumor necrosis factor-alpha (TNF-α) and interferon gamma (IFN-γ) (Figure 1H-M). While HER1-CAR-T cells incubated under the hypoxic condition exhibited significantly reduced secretion of these tumor suppression cytokines. Consistently, con-CAR-T cells with same treatments showed limited cytokine secretion. In short, these results demonstrate that hypoxia condition could do suppress the cell viability, targeted cell killing and effector cytokine secretion capabilities of HER1-CAR-T cells as those previous reports [32].

TMP can activate VEGF expression and eNOS/NO axis

Inspired by the high potency of TMP in remodeling blood vessels through activating the VEGF expression and the eNOS/NO axis inside vascular endothelial cells [33], we therefore explored the possibility of using TMP to attenuate hypoxia and thus benefit HER1-CAR-T therapy against TNBC. Firstly, via the CCK-8 assay, it was shown that commercial TMP molecules at a high incubation concentration of up to ~500 nM imposed insignificant cytotoxicity toward human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC), both of which were utilized as the model target cells of TMP molecules (Figure 2A-B). Then, the potency of TMP treatment in activating the VEGF expression and the eNOS/NO axis inside vascular endothelial cells was carefully investigated via a series of standard assays. Via Western Blotting assay, it was shown that treatment of TMP incubation (500 nM, 24 h) resulted in significantly increased expression of VEGF inside both HUVEC and HAEC cells (Figure 2C-D). It was further uncovered that such TMP incubation promoted the phosphorylation of eNOS, but not obviously impaired the expression of total eNOS (Figure 2E-F). In addition, such TMP treatment was shown to be capable of promoting the release of NO from HUVEC and HAEC cells by using a commercial NO detection kit (Figure 2G). These results collectively demonstrate that TMP treatment can not only enhance VEGF expression, but also promote NO production from vascular endothelial cells (Figure 2H).

Preparation of TMP@PEGgel and its TME modulation effect

The potency of TMP treatment in promoting in vivo NO production was carefully investigated (Figure 3A). To enable sustained release of TMP inside tumors, a type of endogenous H₂O₂ responsive PEGgel was first developed for intratumoral fixation of TMP. It was found that PEGDMA monomer could form hydrogel within one minute upon being added with H₂O₂ and FeCl₂ at optimal feeding ratios (Figure S1A), both of which can undergo Fenton reaction to generate highly reactive hydroxyl group (·OH), a widely used initiator of polymerization [34, 35]. As revealed by scanning electron microscopy, the obtained PEGgel exhibited microscopic porous network structure (Fig. S1B). By recording the absorbance of TMP peaked at 294 nm, it was shown that the TMP loaded PEGgel (TMP@PEGgel) would enable sustained release of TMP (Figure S1C-E). Then, rhodamine B was used as a model fluorophore for tracking the in vivo behavior of non-fluorescent TMP. As visualized under confocal microscopy, obvious rhodamine B fluorescence was observed on the tumor slices collected from mice with intratumoral fixation of rhodamine B with such PEGgel at 3 days and 10 days post injection (p.i.) (Figure 3B and S2). In marked contrast, only obvious rhodamine B fluorescence was observed on the tumors slices for mice collected at 3 days p.i. Therefore, these results indicate that PEGgel could enable prolonger retention of loaded small molecules inside tumors.

Then, the potency of intratumoral TMP fixation on NO production was carefully evaluated on mice bearing MDA-MB-468 and MDA-MB-231 tumors. It was shown that treatment with TMP@PEGgel (TMP = 1 mg/kg) contributed to significantly increased NO production at 3 days and 10 days p.i. compared to these tumor bearing mice with intratumoral injection of saline and plain PEGgel (Figure 3C-D). In marked contrast, the tumor bearing mice with free TMP treatment only led to increased intratumoral NO production at 3 days p.i. Inspired by the potency of VEGF and NO in promoting angiogenesis and maturation of tumor blood vessels [36, 37], we carefully investigated the capacity of TMP@PEGgel treatment in remodeling tumor vasculature. It was shown that treatments with free TMP and TMP@PEGgel were effective in increasing tumor blood vessel intensities in both TNBC xenografts at 3 days p.i., as indicated by the increased expression of CD31, a biomarker of blood vessel endothelial cells, on their tumor slices through the immunofluorescence assay. Furthermore, these two treatments were also be able to increase the percentage of effective blood vessels as indicated by measuring the complete margin of blood vessels at 3 days p.i. (Figure 3E-J). However, only TMP@PEGgel treatment was able to increase intratumoral blood vessel densities and effective vessels percentages at 10 days p.i. In was further shown that plain PEGgel treatment exhibited negligible disturbance on the tumor vasculature. Therefore, these results collectively demonstrate that TMP@PEGgel treatment is capable of remodeling tumor vasculature.

Considering tumor vasculature normalization can enhance tumor blood perfusion and thus tumor oxygenation status, the potency of TMP@PEGgel treatment in relieving tumor hypoxia was therefore carefully evaluated on aforementioned two TNBC tumors xenografts. By using commercial pimonidazole as an exogenous hypoxia-specific probe, we found that treatment with TMP@PEGgel led to dramatically suppressed pimonidazole specific fluorescence signals on tumor slices collected from the these two TNBC tumor bearing mice at 3 days and 10 days p.i. under the microscopic observation (Figure 3K-L and S3A-B). Meanwhile, treatment with free TMP only led to suppressed pimonidazole specific fluorescence on tumor slices collected at 3 days p.i., while other treatments negligibly disturbed the pimonidazole specific fluorescence on tumor slices collected at both time intervals. Moreover, tumor slices of mice with free TMP and TMP@PEGgel treatments showed remarkably reduced expression of hypoxia-inducible factor (HIF)-1α, following similar evolution trends to the results of aforementioned pimonidazole staining assay, via the standard immunofluorescence staining assay (Figure 3M-N and S3C-D). These results indicate that treatment of TMP@PEGgel enabling sustained release of TMP can effectively attenuate tumor hypoxia by remodeling tumor vasculature in these TNBC tumor xenografts.

TMP@PEGgel enhances tumor infiltration, survival time and effector functions of HER1-CAR-T cells

We then investigated the potency of TMP@PEGgel treatment in promoting the tumor infiltration and survival of sequentially administrated HER1-CAR-T cells in both MDA-MB-468 and MDA-MB-231 tumors. HER1-CAR-T cells were first genetically engineered with red fluorescent protein (RFP) for tracking their in vivo behaviors post different treatments. We found that tumor slices collected from mice with intratumoral fixation of TMP (TMP = 1 mg/kg, at day 0) and sequential RFP expressing HER1-CAR-T cells injection (5 × 10⁶ cells, at day 1) exhibited obvious RFP fluorescence signals at day 3 and 10 under the microscopic observation (Figure 4A-C). Consistently, treatment with free TMP only led to obviously increased tumor infiltration of RFP expressing HER1-CAR-T cells at day 3, while other treatments exhibited minimal effects on the tumor infiltration capacity of these systemically administrated RFP expressing HER1-CAR-T. Furthermore, the potency of TMP@PEGgel in promoting tumor infiltration and survival of HER1-CAR-T cells were confirmed using flow cytometry (Figure 4D-E).

We then investigated the effects of TMP@PEGgel treatment on the capacity of tumor-infiltrating HER1-CAR-T cells in secreting effector cytokines (e.g., TNFα, IFN-γ, IL-2), which are tightly associated with the activation of CAR-T cells, by using corresponding enzyme linked immunosorbent assay (ELISA) kits. By following the aforementioned therapeutic regime, it was shown that both TNBC tumors on mice with TMP@PEGgel fixation and sequential HER1-CAR-T cells administration exhibited significantly increased secretion levels of IL-2, TNF-α and IFN-γ within 10 days p.i. (Figure 4F-K). Meanwhile, sequential treatment with free TMP injection and HER1-CAR-T cells administration only led to remarkably increased intratumoral secretion of IL-2, TNF-α and IFN-γ at day 3, while minimally disturbed their secretion at day 10. In sharp contrast, other treatments showed limited impacts on the secretion of these proinflammatory cytokines. These results collectively suggest that TMP@PEGgel treatment can improve the effector functions of HER1-CAR-T cells by enhancing their capacity in secreting proinflammatory cytokines.

TMP@PEGgel enhances the tumor suppression effect of HER1-CAR-T cells

We then assessed the efficacy of TMP@PEGgel treatment in enhancing the in vivo tumor suppression effects of HER1-CAR-T cells on both both TNBC tumor bearing mice as aforementioned. 30 mice bearing MDA-MB-468 tumors were randomly divided into six groups and received following treatments: 1) PBS, 2) con-CAR-T injection, 3) HER1-CAR-T injection, 4) PEGgel fixation + HER1-CAR-T injection, 5) TMP injection + HER1-CAR-T injection, 6) TMP@PEGgel fixation + HER1-CAR-T injection (Figure 5A). TMP (1 mg/kg) in the presence and absence of PEGgel were intratumorally injected at day 0, while HER-1-CAR-T cells or non-CAR-T cells (5 × 10⁶ cells per injection) were intravenous injected for three time at day 1, 2 and 3. By measuring tumor sizes, treatment with sequential TMP@PEGgel fixation and HER1-CAR-T injection exhibited the highest potency in regressing the growth of MDA-MB-468 tumors, 1 tumor completely disappeared while the other four tumors showed no obvious growth within 63 days post the treatment tumors, (Figure 5B). In marked contrast, other treatments only slightly delayed tumor growth, and all of these mice died within 56 days post the corresponding treatments. In addition, body weights of mice with varying treatments were negligible disturbed throughout the whole monitoring process (Figure S4A). Moreover, the excellent therapeutic potency of such TMP@PEGgel assisted HER1-CAR-T treatment was further confirmed on mice bearing MDA-MB-231 tumors (Figure 5C and S4B). These results collectively indicate that sequential treatment of TMP@PEGgel fixation and HER1-CAR-T cells is effective in suppressing the growth of these HER1 positive TNBC tumors.

In conclusion, we prepared an endogenous H₂O₂ responsive hydrogels composed of PEGDMA and FeCl₂ to enable sustained release of Chinese herbal extracts TMP. Upon intratumoral fixation, the obtained TMP@PEGgel promoted angiogenesis and tumor vasculature maturation through activating the VEGF expression and the eNOS/NO axis of vascular endothelial cells. Resultantly, such TMP@PEGgel treatment enabled effective sustained tumor hypoxia relief and thus was capable of enhancing the tumor infiltration, survival and effector functions of intravenously infused HER1-CAR-T cells inside TNBC tumors inoculated on Balb/c nude mice. It was further shown that our TMP@PEGgel assisted HER1-CAR-T cell therapy could thereby synergically suppress the growth of both MDA-MB-468 and MDA-MB-231 TNBC tumors. In this study, it is highlighted that TME remodeling through reprogramming tumor vasculature with TMP@PEGgel treatment is a promising and robust strategy to reinforce the therapeutic potency of CAR-T cell therapies toward solid tumors.

Acknowledgments

We thank the researchers in Feng's lab and Chen’s lab for academic discussions.

Authors’ Contributions

Conceptualization: Y.L, and L.F. Investigation: Y.L., Y.H., X.L., Y.Z., X.M., and Y.Z. Writing, review and editing: Y.L., and L.F.

Fundings

We acknowledge funding supports from National Natural Science Foundation of China (82103099, 22077093), the National Research Programs from Ministry of Science and Technology (MOST) of China (2021YFF0701800, 2022YFF0706500), the Natural Science Foundation of Jiangsu Province (BK20220110), the Suzhou Key Laboratory of Nanotechnology and Biomedicine, the Collaborative Innovation Center of Suzhou Nano Science and Technology, and the 111 Program from the Ministry of Education of China. We also thank the website of app.Biorender.com for the assistance in creating the illustration figures.

Availability of data and materials

All data generated or analyzed in this study can be obtained from the corresponding authors.

Ethics approval

Nanjing Normal University’s medical ethics committee approved the animal experiments in this study.

Consent for publication

Not applicable.

Competing interests

The authors declare no conflicts of interest.

Author details

1. Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Cancer Institute, Department of Biochemistry, College of Life Science, Nanjing Normal University, Nanjing 210023, P. R. China. 2. Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow University, 199 Ren'ai Road, Suzhou 215123, P. R. China. 3. Department of Radiology, The First Affiliated Hospital of Soochow University, Suzhou 215006, P. R. China. 4. Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou, 215000, P. R. China.

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Scheme 1 is available in the Supplementary Files section

No competing interests reported.

SupportingFigures.docx
Scheme1.jpg
A schematic diagram of TMP@PEGgels enhanced HER1-CAR-T cell therapy against TNBC tumors. Upon intratumoral fixation, TME@PEGgel can gradually release TMP to remodel tumor vasculature through simultaneously activating VEGF expression and eNOS/NO axis inside vascular endothelial cells and thus effectively attenuate tumor hypoxia. As a result, TME@PEGgel treatment can promote the tumor infiltration, survival time and effector functions of intravenously injected HER1-CAR-T cells inside TNBC, thereby remarkably reinforcing their tumor suppression effects.

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A Tetramethylpyrazine Releasing Hydrogel can Potentiate CAR-T Cell Therapy against Triple negative breast cancer by Reprogramming Tumor Vasculatures

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