Primary Human Breast Cancer-Associated Endothelial Cells Favor Interactions with Nanomedicines.

Cancer nanomedicines predominately rely on transport processes controlled by tumor-associated endothelial cells to deliver therapeutic and diagnostic payloads into solid tumors. While the dominant role of this class of endothelial cells for nanoparticle transport and tumor delivery is established in animal models, the translational potential in human cells needs exploration. Using primary human breast cancer as a model, the differential interactions of normal and tumor-associated endothelial cells with clinically relevant nanomedicine formulations are explored and quantified. Primary human breast cancer-associated endothelial cells exhibit up to ≈2 times higher nanoparticle uptake than normal human mammary microvascular endothelial cells. Super-resolution imaging studies reveal a significantly higher intracellular vesicle number for tumor-associated endothelial cells, indicating a substantial increase in cellular transport activities. RNA sequencing and gene expression analysis indicate the upregulation of transport-related genes, especially motor protein genes, in tumor-associated endothelial cells. Collectively, the results demonstrate that primary human breast cancer-associated endothelial cells exhibit enhanced interactions with nanomedicines, suggesting a potentially significant role for these cells in nanoparticle tumor delivery in human patients. Engineering nanoparticles that leverage the translational potential of tumor-associated endothelial cell-mediated transport into human solid tumors may lead to the development of safer and more effective clinical cancer nanomedicines.

Optical coherence tomography for multicellular tumor spheroid category recognition and drug screening classification via multi-spatial-superficial-parameter and machine learning.

Optical coherence tomography (OCT) is an ideal imaging technique for noninvasive and longitudinal monitoring of multicellular tumor spheroids (MCTS). However, the internal structure features within MCTS from OCT images are still not fully utilized. In this study, we developed cross-statistical, cross-screening, and composite-hyperparameter feature processing methods in conjunction with 12 machine learning models to assess changes within the MCTS internal structure. Our results indicated that the effective features combined with supervised learning models successfully classify OVCAR-8 MCTS culturing with 5,000 and 50,000 cell numbers, MCTS with pancreatic tumor cells (Panc02-H7) culturing with the ratio of 0%, 33%, 50%, and 67% of fibroblasts, and OVCAR-4 MCTS treated by 2-methoxyestradiol, AZD1208, and R-ketorolac with concentrations of 1, 10, and 25 µM. This approach holds promise for obtaining multi-dimensional physiological and functional evaluations for using OCT and MCTS in anticancer studies.

N-dihydrogalactochitosan reduces mortality in a lethal mouse model of SARS-CoV-2.

The rapid emergence and global dissemination of SARS-CoV-2 that causes COVID-19 continues to cause an unprecedented global health burden resulting in nearly 7 million deaths. While multiple vaccine countermeasures have been approved for emergency use, additional treatments are still needed due to sluggish vaccine rollout, vaccine hesitancy, and inefficient vaccine-mediated protection. Immunoadjuvant compounds delivered intranasally can guide non-specific innate immune responses during the critical early stages of viral replication, reducing morbidity and mortality. N-dihydrogalactochitosan (GC) is a novel mucoadhesive immunostimulatory polymer of ß-0-4-linked N-acetylglucosamine that is solubilized by the conjugation of galactose glycans with current applications as a cancer immunotherapeutic. We tested GC as a potential countermeasure for COVID-19. GC was well-tolerated and did not produce histopathologic lesions in the mouse lung. GC administered intranasally before and after SARS-CoV-2 exposure diminished morbidity and mortality in humanized ACE2 receptor expressing mice by up to 75% and reduced infectious virus levels in the upper airway. Fluorescent labeling of GC shows that it is confined to the lumen or superficial mucosa of the nasal cavity, without involvement of adjacent or deeper tissues. Our findings demonstrate a new application for soluble immunoadjuvants such as GC for preventing disease associated with SARS-CoV-2 and may be particularly attractive to persons who are needle-averse.

Single-cell transcriptomics reveals that tumor-infiltrating natural killer cells are activated by localized ablative immunotherapy and share anti-tumor signatures induced by immune checkpoint inhibitors.

Rationale: Natural killer (NK) cells provide protective anti-cancer immunity. However, the cancer therapy induced activation gene signatures and pathways in NK cells remain unclear. Methods: We applied a novel localized ablative immunotherapy (LAIT) by synergizing photothermal therapy (PTT) with intra-tumor delivering of the immunostimulant N-dihydrogalactochitosan (GC), to treat breast cancer using a mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT) mouse model. We performed single-cell RNA sequencing (scRNAseq) analysis to unveil the cellular heterogeneity and compare the transcriptional alterations induced by PTT, GC, and LAIT in NK cells within the tumor microenvironment (TME). Results: ScRNAseq showed that NK subtypes, including cycling, activated, interferon-stimulated, and cytotoxic NK cells. Trajectory analysis revealed a route toward activation and cytotoxicity following pseudotime progression. Both GC and LAIT elevated gene expression associated with NK cell activation, cytolytic effectors, activating receptors, IFN pathway components, and cytokines/chemokines in NK subtypes. Single-cell transcriptomics analysis using immune checkpoint inhibitor (ICI)-treated animal and human samples revealed that ICI-induced NK activation and cytotoxicity across several cancer types. Furthermore, ICI-induced NK gene signatures were also induced by LAIT treatment. We also discovered that several types of cancer patients had significantly longer overall survival when they had higher expression of genes in NK cells that were also specifically upregulated by LAIT. Conclusion: Our findings show for the first time that LAIT activates cytotoxicity in NK cells and the upregulated genes positively correlate with beneficial clinical outcomes for cancer patients. More importantly, our results further establish the correlation between the effects of LAIT and ICI on NK cells, hence expanding our understanding of mechanism of LAIT in remodeling TME and shedding light on the potentials of NK cell activation and anti-tumor cytotoxic functions in clinical applications.

Immune modulations of the tumor microenvironment in response to phototherapy.

The tumor microenvironment (TME) promotes pro-tumor and anti-inflammatory metabolisms and suppresses the host immune system. It prevents immune cells from fighting against cancer effectively, resulting in limited efficacy of many current cancer treatment modalities. Different therapies aim to overcome the immunosuppressive TME by combining various approaches to synergize their effects for enhanced anti-tumor activity and augmented stimulation of the immune system. Immunotherapy has become a major therapeutic strategy because it unleashes the power of the immune system by activating, enhancing, and directing immune responses to prevent, control, and eliminate cancer. Phototherapy uses light irradiation to induce tumor cell death through photothermal, photochemical, and photo-immunological interactions. Phototherapy induces tumor immunogenic cell death, which is a precursor and enhancer for anti-tumor immunity. However, phototherapy alone has limited effects on long-term and systemic anti-tumor immune responses. Phototherapy can be combined with immunotherapy to improve the tumoricidal effect by killing target tumor cells, enhancing immune cell infiltration in tumors, and rewiring pathways in the TME from anti-inflammatory to pro-inflammatory. Phototherapy-enhanced immunotherapy triggers effective cooperation between innate and adaptive immunities, specifically targeting the tumor cells, whether they are localized or distant. Herein, the successes and limitations of phototherapy combined with other cancer treatment modalities will be discussed. Specifically, we will review the synergistic effects of phototherapy combined with different cancer therapies on tumor elimination and remodeling of the immunosuppressive TME. Overall, phototherapy, in combination with other therapeutic modalities, can establish anti-tumor pro-inflammatory phenotypes in activated tumor-infiltrating T cells and B cells and activate systemic anti-tumor immune responses.

Localized ablative immunotherapy drives de novo CD8+ T-cell responses to poorly immunogenic tumors.

BACKGROUND: Localized ablative immunotherapies hold great promise in stimulating antitumor immunity to treat metastatic and poorly immunogenic tumors. Tumor ablation is well known to release tumor antigens and danger-associated molecular patterns to stimulate T-cell immunity, but its immune stimulating effect is limited, particularly against metastatic tumors. METHODS: In this study, we combined photothermal therapy with a potent immune stimulant, N-dihydrogalactochitosan, to create a local ablative immunotherapy which we refer to as laser immunotherapy (LIT). Mice bearing B16-F10 tumors were treated with LIT when the tumors reached 0.5 cm3 and were monitored for survival, T-cell activation, and the ability to resist tumor rechallenge. RESULTS: We found that LIT stimulated a stronger and more consistent antitumor T-cell response to the immunologically 'cold' B16-F10 melanoma tumors and conferred a long-term antitumor memory on tumor rechallenge. Furthermore, we discovered that LIT generated de novo CD8+ T-cell responses that strongly correlated with animal survival and tumor rejection. CONCLUSION: In summary, our findings demonstrate that LIT enhances the activation of T cells and drives de novo antitumor T-cell responses. The data presented herein suggests that localized ablative immunotherapies have great potential to synergize with immune checkpoint therapies to enhance its efficacy, resulting in improved antitumor immunity.

Single-cell RNA sequencing reveals localized tumour ablation and intratumoural immunostimulant delivery potentiate T cell mediated tumour killing.

BACKGROUND: Metastatic breast cancer poses great challenge in cancer treatment. N-dihydrogalactochitosan (GC) is a novel immunoadjuvant that stimulates systemic immune responses when administered intratumourally following local tumour ablation. A combination of photothermal therapy (PTT) and GC, referred to as localized ablative immunotherapy (LAIT), extended animal survival and generates an activated B cell phenotype in MMTV-PyMT mouse mammary tumour microenvironment (TME). However, how T cell populations respond to LAIT remains to be elucidated. METHODS: Using depletion antibodies, we studied the contributions of CD8+ and CD4+ T cells to the therapeutic effect of LAIT. Using single-cell RNA-sequencing (scRNAseq), we analysed tumour-infiltrating T cell heterogeneity and dissected their transcriptomes upon treatments of PTT, GC, and LAIT (PTT+GC). RESULTS: Loss of CD8+ T cells after LAIT abrogated the therapeutic benefits of LAIT. Ten days after treatment, proportions of CD8+ and CD4+ T cells in untreated TME were 19.2% and 23.0%, respectively. Upon LAIT, both proportions were increased to 25.5% and 36.2%, respectively. In particular, LAIT increased the proportions of naïve and memory cells from a resting state to an activated state. LAIT consistently induced the expression of co-stimulatory molecules, type I IFN responsive genes, and a series of antitumor cytokines, Ifng, Tnf, Il1, and Il17 in CD8+ and CD4+ T cells. LAIT also induced immune checkpoints Pdcd1, Ctla4, and Lag3 expression, consistent with T cell activation. Relevant to clinical translation, LAIT also upregulated genes in CD8+ and CD4+ T cells that positively correlated with extended survival of breast cancer patients. CONCLUSIONS: Overall, our results reveal that LAIT prompts immunological remodelling of T cells by inducing broad proinflammatory responses and inhibiting suppressive signalling to drive antitumour immunity.

Iron oxide nanoparticles as a drug carrier reduce host immunosuppression for enhanced chemotherapy.

Chemotherapy is still regarded as the main modality for cancer treatment. However, it often suppresses the host immune system, resulting in limited therapeutic effects. It is desirable to design a novel chemotherapeutic agent to reduce the level of immunosuppression. Herein, we designed bovine serum albumin (BSA)-bioinspired iron oxide nanoparticles (IONPs) as a nanocarrier to load anticancer drug mitoxantrone (MTX) for enhanced chemotherapy of orthotopic breast cancer. The treatment with IONPs@BSA-MTX complexes increased CD3+CD4+ and CD3+CD8+ T lymphocytes more than free MTX. The complexes effectively restored the host immune system and exhibited a better anticancer efficacy than free MTX. It was worth noting that the BSA-inspired IONPs were a satisfactory contrast agent for magnetic resonance imaging of tumors and lymph nodes. Our work provides a novel strategy for enhanced chemotherapy with low levels of immunosuppression in the treatment of breast cancer and other cancers.

Doxorubicin-Loaded Metal-Organic Framework Nanoparticles as Acid-Activatable Hydroxyl Radical Nanogenerators for Enhanced Chemo/Chemodynamic Synergistic Therapy.

Doxorubicin (DOX) is a widely used first-line antitumor agent; however, acquired drug resistance and side effects have become the main challenges to effective cancer therapy. Herein, DOX is loaded into iron-rich metal-organic framework/tannic acid (TA) nanocomplex to form a tumor-targeting and acid-activatable drug delivery system (MOF/TA-DOX, MTD). Under the acidic tumor microenvironment, MTD simultaneously releases DOX and ferrous ion (Fe2+) accompanied by degradation. Apart from the chemotherapeutic effect, DOX elevates the intracellular H2O2 levels through cascade reactions, which will be beneficial to the Fenton reaction between the Fe2+ and H2O2, to persistently produce hydroxyl radicals (•OH). Thus, MTD efficiently mediates chemodynamic therapy (CDT) and remarkably enhances the sensitivity of chemotherapy. More encouragingly, the cancer cell killing efficiency of MTD is up to ~86% even at the ultralow equivalent concentration of DOX (2.26 µg/mL), while the viability of normal cells remained >88% at the same concentration of MTD. Taken together, MTD is expected to serve as drug-delivery nanoplatforms and •OH nanogenerators for improving chemo/chemodynamic synergistic therapy and reducing the toxic side effects.

Antigen presentation and interferon signatures in B cells driven by localized ablative cancer immunotherapy correlate with extended survival.

Rationale: B cells have emerged as key regulators in protective cancer immunity. However, the activation pathways induced in B cells during effective immunotherapy are not well understood. Methods: We used a novel localized ablative immunotherapy (LAIT), combining photothermal therapy (PTT) with intra-tumor delivery of the immunostimulant N-dihydrogalactochitosan (GC), to treat mice bearing mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT). We used single-cell RNA sequencing to compare the transcriptional changes induced by PTT, GC and PTT+GC in B cells within the tumor microenvironment (TME). Results: LAIT significantly increased survival in the tumor-bearing mice, compared to the treatment by PTT and GC alone. We found that PTT, GC and PTT+GC increased the proportion of tumor-infiltrating B cells and induced gene expression signatures associated with B cell activation. Both GC and PTT+GC elevated gene expression associated with antigen presentation, whereas GC elevated transcripts that regulate B cell activation and GTPase function and PTT+GC induced interferon response genes. Trajectory analysis, where B cells were organized according to pseudotime progression, revealed that both GC and PTT+GC induced the differentiation of B cells from a resting state towards an effector phenotype. The analyses confirmed upregulated interferon signatures in the differentiated tumor-infiltrating B cells following treatment by PTT+GC but not by GC. We also observed that breast cancer patients had significantly longer survival time if they had elevated expression of genes in B cells that were induced by PTT+GC therapy in the mouse tumors. Conclusion: Our findings show that the combination of local ablation and local application of immunostimulant initiates the activation of interferon signatures and antigen-presentation in B cells which is associated with positive clinical outcomes for breast cancer. These findings broaden our understanding of LAIT's regulatory roles in remodeling TME and shed light on the potentials of B cell activation in clinical applications.

Synergistic interventional photothermal therapy and immunotherapy using an iron oxide nanoplatform for the treatment of pancreatic cancer.

Pancreatic cancer (PC) is the most lethal malignancy due to its high metastatic ability and poor drug permeability. Here, a synergized interventional photothermal-immunotherapy strategy was developed with imaging guidance and temperature monitoring by magnetic resonance imaging (MRI) technique, for the local treatment of metastatic PC. A tumor microenvironment (TME)-responsive nanoplatform was fabricated via coating of DSPE-PEG and indocyanine green (ICG) onto imiquimod (IMQ) loaded amorphous iron oxide nanoparticles (IONs). This unique nanoplatform, IMQ@IONs/ICG, served as a contrast agent for MRI, a drug delivery vehicle for IMQ and ICG, and a catalyst for TME modulation. The biodegradable IMQ@IONs/ICG was also non-toxic, and improved the penetration of the loaded drugs in PC to maximize thermal ablation of the tumor and minimize damage to the surrounding healthy tissue. For the treatment of aggressive, metastatic Panc02-H7 pancreatic tumors in mice, ION-assisted MRI was employed to guide the administration of interventional photothermal therapy (IPTT) and monitor the temperature distribution in target tumor and surrounding tissue during treatment. The local IPTT treatment induced in situ immunogenic cell death (ICD), and, in combination with released IMQ, triggered a strong antitumor immunity, leading to decreased metastases and increased CD8+ in spleen and tumors. With precise local treatment and monitoring, treated primary tumors were completely eradicated, mesentery metastases were dramatically reduced, and the survival time was significantly prolonged, without damage to normal tissue and systemic autoimmunity. Overall, this synergistic strategy represents a promising approach to treat PC with significant potential for clinical applications. STATEMENT OF SIGNIFICANCE: Pancreatic cancer (PC) is one of the most lethal malignancies because it is non-permeable to drugs and highly metastatic. In this study, we designed a tumor microenvironment-responsive amorphous iron oxide nanoplatform (ION) to co-deliver photothermal agent (ICG) and toll-like-receptor-7 agonist (IMQ). This biodegradable nanoplatform IMQ@IONs/ICG improved the penetration of the loaded drugs in pancreatic tumor. With MR imaging guidance and temperature monitoring, the precise interventional photothermal therapy on mouse Panc02-H7 orthotopic tumors releases tumor antigens to initiate tumor-special immune responses, amplified by the released IMQ. Our results demonstrate that IMQ@IONs/ICG overcomes the obstacle of drug delivery to pancreatic tumors, and when combined with photothermal therapy, induces a systemic antitumor immunity to control metastatic tumors.

Novel Immune Stimulant Amplifies Direct Tumoricidal Effect of Cancer Ablation Therapies and Their Systemic Antitumor Immune Efficacy.

Ablation therapies have emerged as an effective tool for destroying cancerous tissue, but for advanced and disseminated tumors their application remains mainly a palliative measure. However, it is becoming increasingly clear that this limitation can be redressed by the use of intratumoral immune stimulating agents for amplifying potential antitumor immune responses that are induced by ablation therapies. A novel immune stimulating drug IP-001, a specific variant of the N-dihydrogalactochitosan (GC) family of molecules, has shown to be effective against metastatic tumors, when combined with different forms tumor ablation. It acts as a multi-function immune stimulant both by directly inhibiting cell membrane repair and recycling of ablation-damaged tumor cells, and indirectly by sequestering ablation-released tumor antigens, as well as recruiting and stimulating antigen presenting cells to induce a potent Th1 type T cell response against the cancer. In this review, we briefly discuss the current applications of local ablation for cancer treatment and the effects of GC in combination with other ablation therapies, a therapeutic approach that is pioneering the field of Interventional Immuno-Oncology (IIO).

Transcriptomic analysis of mechanism of melanoma cell death induced by photothermal therapy.

Melanoma is a malignancy with poor prognosis. Its incidence rate has been on the rise and it poses high health and economic challenges to different populations. Photothermal therapy (PTT) served as an effective local therapy in treating various tumors, particularly cutaneous carcinoma like melanoma. To fully understand the mechanisms of tumor cell death induced by PTT, we investigated gene expression and immune cells compositions of B16-F10 tumors after PTT treatment. A total of 256 differentially expressed genes (DEGs) were identified, with 215 being downregulated and 41 upregulated by PTT. Functional annotation showed that most DEGs involved in immune response and inflammatory response. Immune cells compositions inference revealed changes in many immune cells including regulatory T cells, M2 macrophage and B cells after PTT treatment. Our results help delineate the mechanism of cell death at the transcriptional level triggered by non-invasive PTT treatment of melanoma without exogenous light absorbing agents.

Cancer photo-immunotherapy: from bench to bedside.

Targeted therapy and immunotherapy in combination is considered the ideal strategy for treating metastatic cancer, as it can eliminate the primary tumors and induce host immunity to control distant metastases. Phototherapy, a promising targeted therapy, eradicates primary tumors using an appropriate dosage of focal light irradiation, while initiating antitumor immune responses through induced immunogenic tumor cell death. Recently, phototherapy has been employed to improve the efficacy of immunotherapies such as chimeric antigen receptor T-cell therapy and immune checkpoint inhibitors. Phototherapy and immunoadjuvant therapy have been used in combination clinically, wherein the induced immunogenic cell death and enhanced antigen presentation synergy, inducing a systemic antitumor immune response to control residual tumor cells at the treatment site and distant metastases. This review summarizes studies on photo-immunotherapy, the combination of phototherapy and immunotherapy, especially focusing on the development and progress of this unique combination from a benchtop project to a promising clinical therapy for metastatic cancer.

Photo-activated chemo-immunotherapy for metastatic cancer using a synergistic graphene nanosystem.

We developed a novel treatment strategy for metastatic cancer by synergizing photothermal therapy (PTT), chemotherapy, and immunotherapy through a nanosystem to trigger host antitumor immunity. The nanosystem was constructed by loading mitoxantrone (MTX), a chemotherapeutic agent, and SB-431542 (SB), a transforming growth factor beta (TGF-ß) inhibitor, onto reduced graphene oxide (rGO). Intratumoral administration of rGO/MTX/SB, followed by non-invasive irradiation of a near-infrared laser, destroyed local primary tumors and inhibited distant metastases in 4T1 mouse mammary tumor model, which is poorly immunogenic and highly metastatic. After treatment, 70% of the tumor-bearing mice became long-term survivors and developed a tumor type-specific immunity to resist rechallenged tumor cells. We found that rGO-based PTT provided an immunogenic antigen source, forming in situ vaccination with rGO as an immune-adjuvant. The use of SB changed the tumor microenvironment and improved the therapeutic effect of MTX-generated chemotherapy and rGO-based PTT. The immunological functions of MTX, SB, and rGO acted synergistically to induce an effective tumor vaccination, as evidenced by the increased infiltration of tumor-specific cytotoxic CD8+ T lymphocytes and decreased infiltration of regulatory T cells (Tregs) in distal tumors. Collectively, we demonstrated that rGO/MTX/SB combined with laser irradiation provided a synergistic chemo-immuno-photothermal effect against tumors by in situ vaccination and inhibition of immunosuppressive microenvironment. This unique combination embodies a promising approach to treat metastatic cancers by inducing a systemic antitumor response through a local intervention.

Biodegradable pH-responsive amorphous calcium carbonate nanoparticles as immunoadjuvants for multimodal imaging and enhanced photoimmunotherapy.

Development of bioresponsive theranostic nanoparticles to enhance cancer diagnostics and control cancer metastasis is highly desirable. In this study, we developed such a bioresponsive theranostic nanoparticle for synergistic photoimmunotherapy. In particular, these nanoparticles were constructed by embedding indocyanine green (ICG) into Mn2+-doped amorphous calcium carbonate (ACC(Mn)) nanoparticles, followed by loading of the Toll-like-receptor-7 agonist imiquimod (IMQ). The IMQ@ACC(Mn)-ICG/PEG nanoparticles respond to the acidic pH of the tumor microenvironment (TME) and co-deliver ICG and IMQ into the tumor. Selective phototherapy was achieved upon activation using a near-infrared laser. In the presence of IMQ and arising from phototherapeutically treated tumor cells, tumor-associated antigens give rise to a strong antitumor immune response. Reversal of the immunosuppressive TME via H+ scavenging of the tumor through ACC nanoparticles effectively inhibits tumor metastases. Moreover, the combination of ICG and Mn2+ also serves as an advanced contrast agent for cancer multimode imaging. Overall, these bioresponsive nanoparticles provide a promising approach for cancer theranostics with promising potential for future clinical translation.

Immunologically modified MnFe2O4 nanoparticles to synergize photothermal therapy and immunotherapy for cancer treatment.

Immunotherapy has been a promising candidate for cancer treatment. The combination of photothermal therapy (PTT) and immunotherapy have shown to cause tumor ablation and induce host immune response. However, this strategy is often hampered by a limited immune response and undesirable immunosuppression. In this work, we developed an immunologically modified nanoplatform, using ovalbumin (OVA)-coated PEGylated MnFe2O4 nanoparticles (NPs) loaded with R837 immunoadjuvant (R837-OVA-PEG-MnFe2O4 NPs) to synergize PTT and immunotherapy for the treatment of breast cancer. The designed R837-OVA-PEG-MnFe2O4 NPs are able to elicit significant immune responses in vitro and in vivo. MnFe2O4 NPs also allowed for a reduction of systemic immunosuppression through downregulation of M2-associated cytokines. More importantly, the R837-OVA-PEG-MnFe2O4 NPs under laser irradiation effectively inhibited tumor growth and prevented lung metastases, leading to a prolonged survival time and improved survival rate. In addition, the designed multitasking MnFe2O4 NPs showed as a good contrast agent for magnetic resonance (MR) imaging to detect orthotopic breast tumor in vivo. Our work provides a novel strategy for combined PTT and improved immunotherapy in the treatment of breast and other metastatic cancers.

Neutrophil infiltration and whole-cell vaccine elicited by N-dihydrogalactochitosan combined with NIR phototherapy to enhance antitumor immune response and T cell immune memory.

Melanoma is one of the deadliest malignancies with a high risk of relapse and metastasis. Long-term, tumor-specific, and systemic immunity induced by local intervention is ideal for personalized cancer therapy. Laser immunotherapy (LIT), a combination of local irradiation of laser and local administration of an immunostimulant, was developed to achieve such an immunity. Although LIT showed promising efficacy on tumors, its immunological mechanism is still not understood, especially its spatio-temporal dynamics. Methods: In this study, we investigated LIT-induced immunological responses using a 980-nm laser and a novel immunostimulant, N-dihydrogalactochitosan (GC). Then we followed the functions of key immune cells spatially and temporally using intravital imaging and immunological assays. Results: Immediately after LIT, GC induced a rapid infiltration of neutrophils which ingested most GC in tumors. The cytokines released to the serum peaked at 12 h after LIT. Laser irradiations produced photothermal effects to ablate the tumor, release damage-associated molecular patterns, and generate whole-cell tumor vaccines. LIT-treated tumor-bearing mice efficiently resisted the rechallenged tumor and prevented lung metastasis. Intravital imaging of tumor at rechallenging sites in LIT-treated mice revealed that the infiltration of tumor-infiltrating lymphocytes (TILs) increased with highly active motility. Half of TILs with arrest and confined movements indicated that they had long-time interactions with tumor cells. Furthermore, LIT has synergistic effect with checkpoint blockade to improve antitumor efficacy. Conclusion: Our research revealed the important role of LIT-induced neutrophil infiltration on the in situ whole-cell vaccine-elicited antitumor immune response and long-term T cell immune memory.

A pipeline for identification and validation of tumor-specific antigens in a mouse model of metastatic breast cancer.

Cancer immunotherapy continues to make headway as a treatment for advanced stage tumors, revealing an urgent need to understand the fundamentals of anti-tumor immune responses. Noteworthy is a scarcity of data pertaining to the breadth and specificity of tumor-specific T cell responses in metastatic breast cancer. Autochthonous transgenic models of breast cancer display spontaneous metastasis in the FVB/NJ mouse strain, yet a lack of knowledge regarding tumor-bound MHC/peptide immune epitopes in this mouse model limits the characterization of tumor-specific T cell responses, and the mechanisms that regulate T cell responses in the metastatic setting. We recently generated the NetH2pan prediction tool for murine class I MHC ligands by building an FVB/NJ H-2q ligand database and combining it with public information from six other murine MHC alleles. Here, we deployed NetH2pan in combination with an advanced proteomics workflow to identify immunogenic T cell epitopes in the MMTV-PyMT transgenic model for metastatic breast cancer. Five unique MHC I/PyMT epitopes were identified. These tumor-specific epitopes were confirmed to be presented by the class I MHC of primary MMTV-PyMT tumors and their T cell immunogenicity was validated. Vaccination using a DNA construct encoding a truncated PyMT protein generated CD8 + T cell responses to these MHC class I/peptide complexes and prevented tumor development. In sum, we have established an MHC-ligand discovery pipeline in FVB/NJ mice, identified and tracked H-2Dq/PyMT neoantigen-specific T cells, and developed a vaccine that prevents tumor development in this metastatic model of breast cancer.

N-dihydrogalactochitosan-supported tumor control by photothermal therapy and photothermal therapy-generated vaccine.

Photothermal therapy (PTT) is recently clinically established cancer therapy that uses near-infrared light for thermal ablation of solid tumors. The biopolymer N-dihydrogalactochitosan (GC) was shown in multiple reports to act as a very effective adjunct to tumor PTT. In the present study, mouse tumor model SCCVII (squamous cell carcinoma) was used with two protocols, in situ tumor PTT and therapeutic PTT vaccine for tumors, for investigating the effects of GC. The results reveal that GC can potentiate tumoricidal action of PTT through both direct and indirect mechanisms. In addition to previously known capacity of GC for activating immune effector cells, the indirect means is shown to include reducing the populations of immunoregulatory T cells (Tregs) in PTT-treated tumors. Testing the effects of GC on PTT-treated SCCVII tumor cells in vitro uncovered the existence of a direct mechanism evident by reduced colony survival of these cells. Fluorescence microscopy demonstrated increased binding of fluorescein-labeled GC to PTT-treated compared to untreated SCCVII cells that can be blocked by pre-exposure to annexin V. The results of additional in vitro testing with specific inhibitors demonstrate that these direct mechanisms do not involve the engagement of death surface receptors that trigger extrinsic apoptosis pathway signaling but may be linked to pro-survival activity of caspase-1. Based on the latter, it can be suggested that GC-promoted killing of PTT-treated cells stems from interference of GC bound to damaged membrane components with the repair of these structures that consequently hinders cell survival.