Murine Tumor Models Research Articles

Abstract P006: Partial tumor volume irradiation by 177Lu-PNT6555 induces diverse immune responses in a syngeneic murine tumor model

Abstract Purpose/Objective: Radiopharmaceutical therapy (RPT) enables the systemic delivery of an inherently heterogeneous dose of radiation. The RPT dose heterogeneity is created by a tumor’s non-uniform perfusion and the variation in RPT target expression. Radiation therapy is known to immunomodulate the tumor microenvironment (TME) in a dose-dependent manner. The hypothesis is that RPT with a heterogeneous distribution, delivering a low to high dose gradient in a single TME, will activate multiple dose dependent immune effects that are more effective in promoting priming of adaptive immunity compared to a more homogenous distribution of RPT. Methods: RPT was administered using PNT6555, a fibroblast activation protein alpha targeting agent, and uptake was confirmed with B16 murine melanoma. The B16 cell line is engineered to overexpress mouse fibroblast activation protein alpha (mFAP). The engineered B16 was implanted either alone to form a tumor with 100% mFAP expression (B16-mFAP+) or in a mixture with the parental cell line to create a heterogeneous expression of mFAP (mixed B16-mFAP+/B16-mFAP-). Utilizing the SPECT/CT data and ex vivo biodistribution, image-based dosimetry for 177Lu-PNT6555 was performed using the Monte-Carlo-based RAPID platform. C57BL/6 mice bearing B16-mFAP+ or mixed B16-mFAP+/mFAP- tumors were treated with non-radiolabeled PNT6555, 5, 10, 20 or 40 Gy on day 0. Tumors were harvested on day 7 post-treatment. RT-qPCR was performed to evaluate the type I interferon response and tumor immune susceptibility markers. Mice bearing B16-mFAP+ or mixed B16-mFAP+/mFAP- received a mean tumor dose of 5 Gy 177Lu-PNT6555. Mice were monitored for tumor growth and overall survival. In a separate cohort, tumors and tumor draining lymph nodes (TDLN) were harvested at days 4, 7, and 12 post-injection. Flow cytometry was performed on the tumor to evaluate immune infiltration and TDLN to evaluate dendritic cell and B cell activation. Statistical significance was determined by a one-way ANOVA (flow) or Kaplan-Meier with log-rank testing (therapy). Results: Optimal upregulation of immune-related genes by 177Lu-PNT6555 occurred at 2.5 Gy for Icam, 10 Gy for Ifnβ1, and 20 Gy for H2-K1 (murine gene coding MHCI). A heterogeneous delivery of 5 Gy to mice bearing mixed B16-mFAP+/mFAP- resulted in upregulation of all three genes in a single TME. For the therapy study, a heterogeneous distribution of 5 Gy 177Lu-PNT6555 significantly increased overall survival compared to a homogenous distribution. Flow cytometry revealed a significant increase in dendritic cell activation by CD80 and MHCII expression in the 5 Gy 177Lu-PNT6555 with heterogeneous distribution. Additionally, there was a significant increase in infiltration of CD4+ cells, CD8+ cells, NK cells and B cells as well as preservation of CD8+ cell in the 5 Gy 177Lu-PNT6555 with heterogeneous distribution. Conclusions: These preclinical studies demonstrate the capacity of a heterogeneous dose of RPT to enhance the immunomodulatory effect of RPT and improve overall survival in a monotherapy setting. Citation Format: Maya E. Takashima, Ohyun Kwon, Shin Hye Ahn, Anthony Belanger, Won Jong Jin, Charlotte Sawicki, Paul A. Clark, Tessa Chen, Ria Kumari, Bryan Bednarz, Zachary S. Morris. Partial tumor volume irradiation by 177Lu-PNT6555 induces diverse immune responses in a syngeneic murine tumor model.[abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr P006

Human 3D Lung Cancer Tissue Photothermal Therapy Using Zn- and Co-Doped Magnetite Nanoparticles.

Iron oxide-based nanoparticles are promising materials for cancer thermal therapy and immunotherapy. However, several proofs of concept reported data with murine tumor models that might have limitations for clinical translation. Magnetite is nowadays the most popular nanomaterial, but doping with distinct ions can enhance thermal therapy, namely, magnetic nanoparticle hyperthermia (MNH) and photothermal therapy (PTT). In this study, we used a 3D alveolar reconstructed A549 lung cancer tissue model and investigated the thermal properties, toxicity, and impact of the thermal dose on tissue viability and inflammatory response using magnetite codoped with 40% Zn and 2% Co divalent ions. The ZnCo-doped magnetite nanoparticles are not toxic up to an NP concentration of 30 mg/mL. PTT showed a better heat generation response than MNH under the evaluated conditions, while NP showed a high external photothermal conversion efficiency of ∼1.3 g·L-1·cm-1 at 808 nm. PTT study is carried out at different temperatures, 43 and 47 °C, for 15 min. Tissue viability decreased with increasing thermal dose, while intracelullar ROS levels increased, mitochondrial activity decreased, and active caspase-3 increased, suggesting cell death via apoptosis. Nanoparticles and PTT did not influence the cytokine TNF, IL-10, IL-1B, and IL-12p70. In contrast, IL-6 and IL-8 were triggered by NP and PTT. Increased expression of IL-6 and IL-8 with higher thermal doses is correlated with tissue injury results, suggesting the potential role in activating and attracting immune cells to the site of thermal-mediated tissue injury.

Macrophage-specific in vivo RNA editing promotes phagocytosis and antitumor immunity in mice.

Macrophages play a central role in antitumor immunity, making them an attractive target for gene therapy strategies. However, macrophages are difficult to transfect because of nucleic acid sensors that can trigger the degradation of foreign plasmid DNA. Here, we developed a macrophage-specific editing (MAGE) system by which compact plasmid DNA encoding a CasRx editor can be delivered to macrophages by a poly(β-amino ester) (PBAE) carrier to bypass the DNA sensor and enable RNA editing in vitro and in vivo. We identified a four-arm branched PBAE with 1-(2-aminoethyl)-4-methylpiperazine end-capping (PBAE29) that enables highly efficient macrophage transfection. PBAE29-mediated transfection of cultured macrophages stimulated less inflammatory cytokine production and inflammasome activation compared with traditional lipofectamine or electroporation-mediated plasmid delivery. Transfection efficiency was further improved by delivering CasRx by minicircle plasmid. The MAGE system incorporated a layer of carboxylated-mannan coating to target macrophage mannose receptors and a macrophage-specific promoter for enhanced selectivity. The delivery of CasRx with guide RNA targeting the transcripts for sialic acid-binding immunoglobulin similar to lectin 10 and signal regulatory protein alpha expression resulted in effective protein knockdown, improving macrophage phagocytosis. The MAGE system also showed efficacy in targeting macrophages in vivo, stimulating antitumor immune responses and reducing tumor volume in murine tumor models, including patient-derived pancreatic adenocarcinoma xenografts in humanized mice. In sum, the MAGE system presents a promising platform for in vivo macrophage-specific delivery of RNA editing tools that can be applied as a cancer therapy.

Establishment and characterization of a new mouse gastric carcinoma cell line, MCC

BackgroundThe aim of this study was to establish a primary mouse gastric carcinoma cell line.MethodsGastric adenocarcinoma in the body region was induced in immunocompetent BALB/c mice using N-Methyl-N-nitrosourea and a 2% NaCl solution. Fresh gastric cancer tissue samples were cultured in 1640 medium supplemented with 10% fetal bovine serum for primary culture and subculture. Cellular morphology was assessed via light microscopy, and a cell growth curve was established. Genomic and proteomic analyses were conducted to characterize the molecular features of the cell lines. This cell line demonstrated a 100% success rate in forming subcutaneous tumors in BALB/c mice. By integrating proteomic profiles from clinical gastric cancer patients and the murine subcutaneous tumor model, several molecular targets suitable for preclinical investigation were identified. Trametinib, a MEK inhibitor, was employed as a model compound in our preclinical study.ResultsA novel gastric carcinoma cell line, designated MCC, was established from BALB/c mice. This cell line exhibited a doubling time of approximately 33 h. Genomic and proteomic analyses identified mutations frequently observed in clinical gastric cancer patients, such as Kras, Egfr, and Ccnd3. Additionally, MCC overexpresses proteins, including SLC1A5, MCM6, and ITGA2, which are significantly upregulated in gastric cancer tissues compared to adjacent non-cancerous tissues. The MCC cell line demonstrated stable tumorigenicity in immunocompetent BALB/c mice, forming subcutaneous tumors that closely resemble the proteomic profile of clinical gastric cancer samples. This high concordance facilitated the identification of several potential therapeutic targets for gastric cancer. Preclinical studies with trametinib revealed that treatment effectively inhibited gastric cancer growth, likely mediated through the activation of immune cells, particularly neutrophils and T cells.ConclusionsThe MCC cell line serves as an indispensable model for gastric cancer research, offering a robust platform for investigating tumor development and progression. Its exceptional tumorigenic capacity and strong concordance with clinical proteomic profiles underscore its significance in translational research, facilitating the discovery of novel therapeutic targets and elucidation of molecular pathways critical for developing effective treatment strategies.

Snapshot Imaging of Stokes Vector Polarization Speckle in Turbid Optical Phantoms and In Vivo Tissues

Significance: We present a system to measure and analyze the complete polarization state distribution of speckle patterns generated from in vivo tissue. Accurate measurement of polarization speckle requires both precise spatial registration and rapid polarization state acquisition. A unique measurement system must be designed to achieve accurate images of polarization speckle patterns for detailed investigation of the scattering properties of biological tissues in vivo. Aim and approach: This system features a polarization state analyzer with no moving parts. Two pixel-polarizer cameras allow for the instantaneous acquisition of the spatial Stokes vector distribution of polarization speckle patterns. System design and calibration methods are presented, and representative images from measurements on liquid phantoms (microsphere suspensions) and in vivo healthy and tumor murine models are demonstrated and discussed. Results and Conclusions: Quantitative measurements of polarization speckle from microsphere suspensions with controlled scattering coefficients demonstrate differences in speckle contrast, speckle size, and the degree of polarization. Measurements on in vivo murine skin and xenograft tumor tissue demonstrate the ability of the system to acquire snapshot polarization speckle images in living systems. The developed system can thus rapidly and accurately acquire polarization speckle images from different media in dynamic conditions such as in vivo tissue. This capability opens the potential for future detailed investigation of polarization speckle for in vivo biomedical applications.

Comparative Analysis of Systemic Immune Responses and Metastatic Risks in Tumor Ablation: An Animal Study of Radiofrequency Ablation and Irreversible Electroporation with Immune Modulation.

This study aimed to compare systemic immune responses and metastatic effects induced by radiofrequency ablation (RFA) and irreversible electroporation (IRE) in murine tumor models. We assessed cytokine production, growth of treated and untreated metastatic tumors, and synergy with immune checkpoint inhibitors (ICIs). Hep55.1c murine hepatoma cells were implanted in C57BL/6N mice to establish primary tumors. In Experiment 1 (n = 50), RFA or IRE was applied to primary tumors, followed by CD8+ T cell depletion in some groups to assess anti-tumor immune responses. Experiment 2 (n = 45) tested RFA and IRE combined with anti-PD-1 therapy for enhanced abscopal effects. In Experiment 3 (n = 28), anti-IL-6 antibody was administered in IRE-treated mice to examine IL-6's role in secondary tumor growth. Tumor volumes and cytokine/chemokine levels were monitored. Both techniques induced significant CD8+ T cell-mediated anti-tumor responses, with abscopal effects observed in untreated secondary tumors. CD8+ T cell depletion abolished these effects, confirming their role in systemic tumor control. Anti-PD-1 therapy combination further suppressed secondary tumor growth. However, IRE uniquely elevated IL-6 and other inflammatory cytokines, unexpectedly accelerating secondary tumor growth. Administration of an anti-IL-6 antibody mitigated this effect, reducing secondary tumor progression. The results of this animal study indicate that both techniques promote systemic anti-tumor immunity, though IRE uniquely induces an inflammatory response that risks exacerbating micro-metastases through IL-6. Combining IRE with IL-6 blockade may offer a promising strategy for nonthermal tumor ablation therapies. Further studies are warranted to refine ablation-immune therapy combinations for optimal therapeutic outcomes.

Ultrasound-Guided Histotripsy Triggers the Release of Tumor-Associated Antigens from Breast Cancers.

Background/Objectives: There is increasing evidence to indicate that histotripsy treatment can enhance the host anti-tumor immune responses both locally at the targeting tumor site as well as systemically from abscopal effects. Histotripsy is a non-invasive ultrasound ablation technology that mechanically disrupts target tissue via cavitation. A key factor contributing to histotripsy-induced abscopal effects is believed to be the release of tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs) that induce a systemic immune response. In this study, we studied the effect of histotripsy treatment on the release of HER2, a well-defined TAA target for cancer immunotherapy. Methods: A range of doses of histotripsy administered to HER2-postive mammary tumor cells in an in vitro cell culture system and an ex vivo tumor were applied. In addition, a single dose of histotripsy was used for an in vivo murine tumor model. The released proteins, and specifically HER2, in both tumor cell-free supernatants and tumor cell pellets were analyzed by a BCA protein assay, an ultra-performance liquid chromatography (UPLC) assay, and Western blot. Results: Our results showed that histotripsy could significantly trigger the release of HER2 proteins in the current study. The level of HER2 proteins was actually higher in tumor cell-free supernatants than in tumor cell pellets, suggesting that HER2 was released from the intracellular domain into the extracellular compartment. Furthermore, proportionally more HER2 protein was released at higher histotripsy doses, indicating free HER2 was histotripsy-dose-dependent. Conclusions: In conclusion, we have qualitatively and quantitatively demonstrated that histotripsy treatment triggers the release of HER2 from the tumor cells into the extracellular compartment. The histotripsy-mediated release of HER2 antigens provides important insights into the mechanism underlying its immunostimulation and suggests the potential of TSA/TAA-based immunotherapies in numerous cancer types.

Co-activating STING-TLR9 pathways promotes radiotherapy-induced cancer vaccination.

Vaccination may cure cancer patients by inducing tumor-specific immune responses. Radiotherapy is an appealing strategy to generate cancer vaccines in situ; thus far, however, only modest and short-lived immune responses are achieved. We here show that radiation combined with co-activating STING-TLR9 can generate powerful in situ cancer vaccines. Notably, radiation at a dose of 12Gy is found to be optimal for boosting tumor cell immunogenicity, and STING-TLR9 co-stimulation by a dual immune activation nano-agonist overrides key immunosuppressive effects associated with radiotherapy. Local radiotherapy combined with the dual immune activation nano-agonists elicits strong systemic anti-tumor immune responses, resulting in complete regression of tumors and metastases in multiple syngeneic murine tumor models. This work introduces a novel and highly potent cancer immunotherapeutic strategy that holds promise for the personalized treatment of intractable cancers.

PCBP2-dependent secretion of miRNAs via extracellular vesicles contributes to the EGFR-driven angiogenesis.

Rationale: The EGFR-driven angiogenesis is crucial in solid tumors, particularly through the delivery of biomolecules via extracellular vesicles (EVs), but the mechanism by which EGFR regulates EV cargo is still unclear. Methods: First, cell co-culture and murine tumor models were employed to examine the impact of EGFR overexpression on the pro-angiogenic properties of small EVs (sEVs) derived from oral squamous cell carcinoma (OSCC). Small RNA sequencing was then used to compare the miRNA profiles of OSCC-sEVs with and without EGFR overexpression, followed by functional enrichment and motif analyses of the differentially expressed miRNAs. Next, miRNA pull-down assays were conducted to identify potential molecules involved in sorting these miRNAs. Finally, the role of the candidate sorting protein was validated using existing public database, tissue samples, cell lines, and murine tumor models. Results: EGFR overexpression significantly enhances the pro-angiogenic effects of OSCC-sEVs, accompanied by a marked increase in the content of nucleic acid cargo carried in these sEVs. Small-RNA sequencing identified a group of miRNAs that were significantly enriched in OSCC-sEVs due to EGFR overexpression, which primarily functioned in angiogenesis and shared a characteristic "GGGU" motif. EGFR overexpression also strengthened the binding of PCBP2 with miRNAs containing this "GGGU" motif, thereby promoting their secretion through sEVs to support tumor angiogenesis. Mechanismly, EGFR overexpression upregulates PCBP2 protein content by activating its transcription rather than regulating the mRNA stability in OSCC cells. Additionally, depletion of PCBP2 impaired the EGFR-driven tumor angiogenesis by inhibiting the secretion of pro-angiogenic miRNAs through sEVs. Conclusions: EGFR boosts PCBP2 expression via transcriptional regulation, which then promotes the loading of specific miRNAs into sEVs by binding to the "GGGU" motif, thereby driving tumor angiogenesis.

Facile integration of a binary nano-prodrug with αPD-L1 as a translatable technology for potent immunotherapy of TNBC.

Immune checkpoint blockers (ICBs)-based immunotherapy is a favorable approach for efficient triple-negative breast cancer (TNBC) treatment. However, the therapeutic efficacy of ICBs is greatly compromised by immunosuppressive tumor microenvironments (TMEs) and low expression levels of programmed cell death ligand-1 (PD-L1). Herein, we constructed an amphiphilic prodrug by linking a hydrophobic STING agonist, MSA-2 and a hydrophilic chemotherapeutic drug, gemcitabine (GEM) via an ester bond, which can self-assemble into GEM-MSA-2 (G-M) nanoparticles (NPs) with a tumor growth inhibition (TGI) value of 87.1% in a murine 4T1 transplantation tumor model. Notably, the immunogenic cell death (ICD)-triggering effect of GEM together with the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway activation properties of MSA-2 enables efficient infiltration of non-exhausting T cells and repolarization of macrophages from M2 to M1 types in the tumor microenvironment for transforming a cold tumor to a hot one. Most importantly, G-M NPs treatment increases the PD-L1 expression levels, thus providing a unique opportunity for further integration with anti-PD-L1 monoclonal antibody (αPD-L1) for eliciting stronger immunity that ultimately leads to a TGI value of 98.0% in the primary tumor and significantly protects against distal and disseminated tumor rechallenge. Overall, this study presents a minimalist nano-prodrug combined with αPD-L1 as a simple yet robust translatable nanotechnology for potent chemo-immunotherapy of TNBC. STATEMENT OF SIGNIFICANCE: Enhancing the therapeutic efficacy of αPD-L1 for tumor immunotherapy via a translatable technology remains a challenge. We report herein facile integration of a binary nano-prodrug with αPD-L1 for potent immunotherapy of TNBC. An amphiphilic prodrug is constructed by linking a hydrophobic STING agonist, MSA-2 and a hydrophilic chemotherapeutic drug, gemcitabine (GEM) via an ester bond. The resulting self-assembled GEM-MSA-2 (G-M) nanoparticles (NPs) show a tumor growth inhibition (TGI) value of 87.1% in a murine 4T1 transplantation tumor model. Besides the induced immunogenic cell death (ICD) and activated cGAS-STING pathway, G-M NPs increase the PD-L1 expression levels, providing a unique opportunity for further integration with αPD-L1 to elicit stronger immunity that ultimately leads to a TGI value of 98.0% in the primary tumor and significantly protects against distal and disseminated tumor rechallenge.

Impact of the tumor microenvironment on delivery of nanomedicine in tumors treated with ultrasound and microbubbles.

The delivery of nanoparticles to tumors has been shown preclinically to be improved by microbubble-mediated ultrasound. However, the mechanisms and biological effects not fully understood. In this study, we explored the influence of the tumor microenvironment on nanoparticle uptake and microdistribution both with and without ultrasound and microbubble treatment. Three murine tumor models, KPC (pancreatic ductal adenocarcinoma), 4 T1 (triple negative mammary carcinoma) and CT26 (colon carcinoma), were characterized with respect to extracellular matrix composition, tumor stiffness and perfusion. KPC and 4 T1 tumors presented higher levels of collagen and hyaluronic acid and were stiffer compared to CT26, whereas all three tumors had similar levels of sulfated glycosaminoglycans. Furthermore, the 4 T1 tumors appeared poorly vascularized with a lower cell density compared to KPC and CT26. All three tumors presented similar nanoparticle uptake, but extravasated nanoparticles traveled significantly shorter in KPC tumors compared to 4 T1 and CT26. The effect of ultrasound and microbubble treatment on the tumor uptake and penetration of polymer nanoparticles into the extracellular matrix were evaluated using a treatment protocol previously shown to increase nanoparticle delivery to tumors. Interestingly, we found a significant increase in nanoparticle uptake in the soft CT26 tumor, but no effect of the ultrasound treatment in the stiff KPC and 4 T1 tumors, suggesting that tumor stiffness is an important parameter for treatment with ultrasound and microbubbles. Ultrasound treatment resulted in a modest but not statistically significant improvement in nanoparticle penetration through the extracellular matrix. In tumors demonstrating increased uptake of nanoparticles following ultrasound treatment, the uptake correlated positively with blood volume. These findings emphasize the importance of taking the tumor microenvironment into consideration when optimizing ultrasound parameters for delivery of nanomedicine.

Near Infrared Thermoluminescent Liposome Sensor for Real‐Time Monitoring of Photothermal Therapy and Magnetic Hyperthermia

AbstractLiposomes are established in the clinic as drug delivery system, and can be designed for heat‐triggered drug release. Thermal nanotherapies, like magnetic nanoparticle hyperthermia (MNH) and photothermal therapy (PTT), show great promise for cancer therapy and other diseases, but the great challenge of them is temperature detection. In this study, a thermoluminescent vesicle for real‐time monitoring of PTT and MNH is developed. The temperature sensor is a liposome containing IR780 dyes incorporated in the membrane. For PTT, the liposome is the thermal agent and temperature sensor, while for MNH only the sensor, since the nanoheaters are Mn‐based iron oxide nanoparticles. Mn‐ferrite nanoparticles showed a good response at low field MNH conditions. The photothermal conversion efficiency of the liposomes altered from 11% to 22%. Thermal sensitivity varied from 0.3% to 5.0% and change after freeze‐drying, while repeatability ranged from 0.91 to 0.98. Photostability studies of MNH and PTT with five heat‐cooling cycles confirmed the temperature sensor application. In vivo and post‐mortem studies using the B16F10 murine tumor model are reported. After intratumoral injection of the thermoluminescent vesicle a blue‐like fluorescence peak shift of ≈20 nm is observed, which maintains the thermometry capabilities and reveals remotely the intratumoral temperature during PTT. The results demonstrate that the near‐infrared thermoluminescent liposome is able to real‐time monitoring the thermal therapy.

An Oral Nanovaccine Secreted by Genetically Engineered and Ultrasound‐Responsive Bacteria for Colon Cancer Immunotherapy

AbstractColorectal cancers represent a major global morbidity and mortality burden, neccessitating improved treatment paradigms. In this work, an ingestible, genetically engineered Escherichia coli (E. coli) 1917 termed “E. coli (AH1‐CDA‐Co1)” is designed that upon ultrasound exposure secretes bacterial outer membrane vesicles (OMV) incorporating the AH1 tumor rejection epitope, an enzyme producing the stimulator of interferon genes (STING) agonist CDA, and the microfold cell‐targeting peptide Co1. For oral administration, a polydopamine system (iPDA) coating on bacteria is exploited to resist the acidic condition in stomach, increase the bacterial survival, and prolong the intestinal transit time. Upon harmless ultrasound exposure, sustained secretion of engineered OMV vaccines is triggered that efficiently cross the intestinal epithelium. Both cyclic GMP–AMP synthase (cGAS)‐STING and TLR4 innate immune signaling pathways are activated, triggering long‐term antigen‐specific immune responses that overcome the immunosuppressive tumor microenvironment. In subcutaneous and orthotopic murine colorectal tumor models, the E. coli (AH1‐CDA‐Co1)@iPDA system inhibits tumor growth and prolongs survival without recurrence. E. coli (AH1‐CDA‐Co1)@iPDA also inhibits tumor growth and recurrence in a postoperative orthotopic colonrectal tumor model of lymph node metastases. Taken together, E. coli (AH1‐CDA‐Co1)@iPDA demonstrates a potent oral vaccine system for improved colon cancer immunotherapy.

Molecular MR Imaging of T-Cell Immune Response to Cryoablation in Immunologically Hot vs. Cold Hepatocellular Carcinoma.

Background and aimsThe increasing enthusiasm for integrating locoregional therapy in primary liver cancer with systemic immunotherapy underscores the need for non-invasive imaging biomarkers. This study aims to establish advanced molecular magnetic resonance imaging (MRI) tools for monitoring T-cell responses to cryoablation in murine models, distinguishing between immunologically "hot" and "cold" hepatocellular carcinoma (HCC). Materials and methodsImmunocompetent 7-10-weeks-old C57BL/6J and BALB/cJ mice (n = 18 each) received carbon tetrachloride for 12 weeks to induce liver cirrhosis. Intrinsically immunogenic Hepa1-6 (“hot”) and non-immunogenic TiB75 (“cold”) cells were orthotopically implanted in C57BL/6 or BALB/c mice, respectively, to generate focal HCC lesions. After one week, animals were randomly assigned to (A) partial cryoablation (pCryo) (1.2 mm cryoprobe, -40°C) or (B) no treatment groups (n=8 per group and tumor type). Gadolinium 160 (160Gd)-labeled CD8+ antibody was administered intravenously either one week after tumor induction (control) or one-week post (pCryo) (treatment). T1-weighted MRI scans were performed using a 9.4 Tesla MRI scanner. Radiological-pathological correlation included imaging mass cytometry (IMC) and immunohistochemistry. ResultsPCryo-treated Hepa1-6 tumors displayed peritumoral ring enhancement on T1-weighted MRI with 160Gd-CD8, correlating with IMC signal patterns. Untreated Hepa1-6 tumors lacked such enhancement. Radiological-pathological correlation confirmed significantly increased tumor-infiltrating CD8+ T-lymphocytes in pCryo Hepa1-6 tumors compared with untreated tumors (p < 0.001), and a stronger local response compared with systemic lymph nodes (p=0.0415). Increased T-lymphocyte infiltration was not observed in TiB75 tumors, as indicated by MRI and histopathology. ConclusionPCryo induced increased T-cell infiltration in Hepa1-6 tumors compared to TiB75 tumors. T1-weighted MRI, following 160Gd-CD8 antibody administration, reproducibly detected the ablation-induced changes. These findings encourage further investigation of MR-based molecular imaging biomarkers to assess immune responses in local tumor therapies. Impact and implicationsThis study successfully established reliable MR-based molecular imaging tools to visualize CD8+ anti-tumor specific T-cell infiltration following partial cryoablation (pCryo) in murine tumor models. The study's significance lies in advancing our understanding of immune responses within induced cirrhosis and distinguishing between "hot" and "cold" tumor phenotypes. The findings not only build upon previous proof-of-principle data but also extend this technology to include different immune cell types in hepatocellular carcinoma (HCC). The study reveals that pCryo may exert specific effects on the tumor microenvironment, augmenting the anti-tumor immune response in immunogenic tumors while displaying a weaker local effect in non-immunogenic tumors.

Personalized Membrane Protein Vaccine Based on a Lipid Nanoparticle Delivery System Prevents Postoperative Recurrence in Colorectal Cancer Models

While accessing tumor neoantigens and developing effective delivery systems have posed significant challenges in therapeutic oncology vaccines, this study introduces a cost- and time-efficient personalized tumor vaccine demonstrating potent anti-tumor effects in a mouse xenograft model. This vaccine utilizes a lipid nanoparticle (C5 LNP) system loaded with membrane protein antigens (mAg) derived from surgically excised tumor tissue. Its safety and efficacy were validated in a B16-OVA murine model. C5/OVA exhibited significant uptake by dendritic cells (DCs), leading to cross-presentation, maturation, and subsequent initiation of robust cell-mediated and humoral immune responses. This resulted in clear tumor growth suppression and extended survival in the B16-OVA model. Furthermore, the personalized C5/mAg vaccine effectively inhibited tumor growth in a colorectal carcinoma model. When combined with anti-PD-1 therapy, it notably increased complete remission (CR) rates in the CT26 xenograft model. Vaccinated mice demonstrated 100% resistance to tumor rechallenge, underscoring the vaccine's ability to induce long-term immune memory. This study presents a promising personalized cancer vaccine delivery system with potential for both treatment and prevention of carcinoma in clinical applications. Statement of significanceIn this paper, we present a scheme for manufacturing personalized tumor vaccines. The vaccine component consists of lipid nanoparticles (LNPs) designated C5 and membrane protein antigens (mAg) derived from autologous tumor tissue surgically resected from the patient. Our study demonstrates that the personalized mAg-C5 vaccine for colorectal carcinoma significantly inhibits tumor growth. Furthermore, conjugation with anti-PD-1 therapy demonstrably increased the complete remission (CR) rate in the murine CT26 xenograft tumor model. Additionally, mice treated with the C5/mAg vaccine exhibited 100% resistance to tumor growth in a colon carcinoma rechallenge model, indicating the induction of immune memory by the vaccine.Our research results suggest that the mAg-LNP vaccine is a simple, cost-effective treatment that clinicians can easily and quickly integrate into routine practice.

Phase separation of chimeric antigen receptor promotes immunological synapse maturation and persistent cytotoxicity.

Major challenges of chimeric antigen receptor (CAR)-T cell therapy include poor antigen sensitivity and cell persistence. Here, we report a solution to these issues by exploiting CAR phase separation. We found that incorporation of an engineered Tcell receptor CD3ε motif, EB6I, into the conventional 28Z or BBZ CAR induced self-phase separation through cation-π interactions. EB6I CAR formed a mature immunological synapse with the CD2 corolla to transduce efficient antigen and costimulatory signaling, although its tonic signaling remained low. Functionally, EB6I CAR-T cells exhibited improved signaling and cytotoxicity against low-antigen tumor cells and persistent tumor-killing function. In multiple primary and relapsed murine tumor models, EB6I CAR-T cells exerted better antitumor functions than conventional CAR-T cells against blood and solid cancers. This study thus unveils a CAR engineering strategy to improve CAR-T cell immunity by leveraging molecular condensation and signaling integration.

Tumor-Targeted Cell-Penetrating Peptides Reveal That Monomethyl Auristatin E Temporally Modulates the Tumor Immune Microenvironment.

Chemotherapies remain standard therapy for cancers but have limited efficacy and cause significant side effects, highlighting the need for targeted approaches. In the progression of cancer, tumors increase matrix metalloproteinase (MMP) activity. Leveraging and therapeutically redirecting tumor MMPs through activatable cell-penetrating peptide (ACPP) technology offers new approaches for tumor-selective drug delivery and for studying how drug payloads engage the tumor immune microenvironment. ACPPs are biosensing peptides consisting of a drug-conjugated polycationic cell-penetrating peptide masked by an autoinhibitory polyanionic peptide through an interlinking peptide linker. Since tumors overexpress MMPs, ACPP tumor-targeting is achieved using an MMP cleavable linker. Monomethyl auristatin E (MMAE) is a potent anti-tubulin and common drug payload in antibody drug conjugates; however there are limited pre-clinical studies on how this clinically effective drug modulates the interplay of cancer cells and the immune system. Here, we report the versatility of ACPP conjugates in syngeneic murine cancer models and interrogate how MMAE temporally alters the tumor immune microenvironment. We show that cRGD-ACPP-MMAE preferentially delivered MMAE to tumors in murine models. Targeted cRGD-ACPP-MMAE demonstrated anti-tumor kill activity that activated the innate and adaptive arms of the immune system. Understanding how targeted MMAE engages tumors can optimize MMAE tumor kill activity and inform rational combinations with other cancer therapeutics.

Blocking WNT7A Enhances MHC-I Antigen Presentation and Enhances the Effectiveness of Immune Checkpoint Blockade Therapy.

The limited infiltration of CD8+ T cells in tumors hampers the effectiveness of T cell-based immunotherapy, yet the mechanisms that limit tumor infiltration by CD8+ T cells remain unclear. Through bulk RNA sequencing of human tumors, we identified a strong correlation between WNT7A expression and reduced CD8+ T-cell infiltration. Further investigation demonstrated that inhibiting WNT7A substantially enhanced MHC-I expression on tumor cells. Mechanistically, WNT7A inhibition inactivated Wnt/β-catenin signaling pathway and thus resulted in reduced physical interaction between β-catenin and p65 in the cytoplasm, which increased the nuclear translocation of p65 and activated the NF-κB pathway, ultimately promoting the transcription of genes encoding MHC-I molecules. We found that our lead compound, 1365-0109, disrupted the protein-protein interaction between WNT7A and its receptor FZD5, resulting in the upregulation of MHC-I expression. In murine tumor models, both genetic and pharmaceutical suppression of WNT7A led to increased MHC-I levels on tumor cells, and consequently enhanced the infiltration and functionality of CD8+ T cells, which bolstered antitumor immunity and improved the effectiveness of immune checkpoint blockade therapy. These findings have elucidated the intrinsic mechanisms of WNT7A-induced immune suppression, suggesting that therapeutic interventions targeting WNT7A hold promise for enhancing the efficacy of immunotherapy.

Mechanical forces inducing oxaliplatin resistance in pancreatic cancer can be targeted by autophagy inhibition

Pancreatic cancer remains one of the most lethal malignancies, with limited treatment options and poor prognosis. A common characteristic among pancreatic cancer patients is the biomechanically altered tumor microenvironment (TME), which among others is responsible for the elevated mechanical stresses in the tumor interior. Although significant research has elucidated the effect of mechanical stress on cancer cell proliferation and migration, it has not yet been investigated how it could affect cancer cell drug sensitivity. Here, we demonstrated that mechanical stress triggers autophagy activation, correlated with increased resistance to oxaliplatin treatment in pancreatic cancer cells. Our results demonstrate that inhibition of autophagy using hydroxychloroquine (HCQ) enhanced the oxaliplatin-induced apoptotic cell death in pancreatic cancer cells exposed to mechanical stress. The combined treatment of HCQ with losartan, a known modulator of mechanical abnormalities in tumors, synergistically enhanced the therapeutic efficacy of oxaliplatin in murine pancreatic tumor models. Furthermore, our study revealed that the use of HCQ enhanced the efficacy of losartan to alleviate mechanical stress levels and restore blood vessel integrity beyond its role in autophagy modulation. These findings underscore the potential of co-targeting mechanical stresses and autophagy as a promising therapeutic strategy to overcome drug resistance and increase chemotherapy efficacy.

Liposomal Codelivery of Doxorubicin and Curcumin Sensitizes Antitumor Activity and Reduces Tumor Metastasis.

Multidrug resistance (MDR) is a major obstacle to traditional cancer treatment using chemotherapeutic agents like doxorubicin (DOX). MDR affects drug dosage regimens and enables the recurrence and metastasis of cancer. Because DOX causes severe side effects at high dosages, it is important to use an MDR modulator to make cancer cells sensitive to DOX. This work focused on a liposome-based codelivery system containing curcumin (CUR) and DOX, focusing on CUR as an MDR modulator. The synergistic effect was maximized when the ratio of DOX and CUR was 1:1, and the synthesis of liposomal drugs was successfully verified. In addition, a successful MDR reversal effect was demonstrated through rhodamine 123 assay, Western blotting, and immunofluorescence. Compared to the conventional DOX treatment, the dual-drug treatment exhibits a significantly improved anticancer effect. In the murine metastasis 4T1 IP tumor model, the dual-drug-encapsulating liposomes successfully suppressed tumor growth and reversed the tumoral effect (omental tumor metastasis, fat, and muscle weight loss) into a normal state.