Photoimmunotheranostics of epithelioid sarcoma by targeting CD44 or EGFR.

Epithelioid sarcoma (ES) is a rare soft tissue neoplasm with high recurrence rates. Wide surgical resection remains the only potential curative treatment. ES presents most commonly on the fingers, hands and forearm, making light-based cancer cell-targeted therapies such as near-infrared photoimmunotherapy (NIR-PIT) that is target-specific, but with limited penetration depth, suitable for ES treatment. We established that CD44 and EGFR were overexpressed in ES patient samples and in the VA-ES-BJ human ES cell line. NIR-PIT of VA-ES-BJ cells using antibody photosensitizer conjugates, prepared by conjugating a CD44 or EGFR monoclonal antibody to the photosensitizer IR700, confirmed that NIR-PIT with both conjugates resulted in cell death. Neither treatment with NIR light alone nor treatment with the conjugates but without NIR light were effective. CD44-IR700-PIT resulted in greater cell death than EGFR-IR700-PIT, consistent with the increased expression of CD44 by VA-ES-BJ cells. In tumors, EGFR-IR700 exhibited a higher tumor-to-normal ratio, as determined by in vivo fluorescence imaging, and a higher anti-tumor growth effect, compared to CD44-IR700. No antitumor effect of the EGFR antibody or the photosensitizer conjugate alone was observed in vivo. Our data support evaluating the use of EGFR-IR700-PIT in the management of ES for detecting and eliminating ES cells in surgical margins, and in the treatment of superficial recurrent tumors.

Prostate fibroblasts and prostate cancer associated fibroblasts exhibit different metabolic, matrix degradation and PD-L1 expression responses to hypoxia.

Fibroblasts are versatile cells that play a major role in wound healing by synthesizing and remodeling the extracellular matrix (ECM). In cancers, fibroblasts play an expanded role in tumor progression and dissemination, immunosuppression, and metabolic support of cancer cells. In prostate cancer (PCa), fibroblasts have been shown to induce growth and increase metastatic potential. To further understand differences in the functions of human PCa associated fibroblasts (PCAFs) compared to normal prostate fibroblasts (PFs), we investigated the metabolic profile and ECM degradation characteristics of PFs and PCAFs using a magnetic resonance imaging and spectroscopy compatible intact cell perfusion assay. To further understand how PFs and PCAFs respond to hypoxic tumor microenvironments that are often observed in PCa, we characterized the effects of hypoxia on PF and PCAF metabolism, invasion and PD-L1 expression. We found that under normoxia, PCAFs displayed decreased ECM degradation compared to PFs. Under hypoxia, ECM degradation by PFs increased, whereas PCAFs exhibited decreased ECM degradation. Under both normoxia and hypoxia, PCAFs and PFs showed significantly different metabolic profiles. PD-L1 expression was intrinsically higher in PCAFs compared to PFs. Under hypoxia, PD-L1 expression increased in PCAFs but not in PFs. Our data suggest that PCAFs may not directly induce ECM degradation to assist in tumor dissemination, but may instead create an immune suppressive tumor microenvironment that further increases under hypoxic conditions. Our data identify the intrinsic metabolic, ECM degradation and PD-L1 expression differences between PCAFs and PFs under normoxia and hypoxia that may provide novel targets in PCa treatment.

Metabolic fingerprinting by nuclear magnetic resonance of hepatocellular carcinoma cells during p53 reactivation-induced senescence.

Cellular senescence is characterized by stable cell cycle arrest. Senescent cells exhibit a senescence-associated secretory phenotype that can promote tumor progression. The aim of our study was to identify specific nuclear magnetic resonance (NMR) spectroscopy-based markers of cancer cell senescence. For metabolic studies, we employed murine liver carcinoma Harvey Rat Sarcoma Virus (H-Ras) cells, in which reactivation of p53 expression induces senescence. Senescent and nonsenescent cell extracts were subjected to high-resolution proton (1H)-NMR spectroscopy-based metabolomics, and dynamic metabolic changes during senescence were analyzed using a magnetic resonance spectroscopy (MRS)-compatible cell perfusion system. Additionally, the ability of intact senescent cells to degrade the extracellular matrix (ECM) was quantified in the cell perfusion system. Analysis of senescent H-Ras cell extracts revealed elevated sn-glycero-3-phosphocholine, myoinositol, taurine, and creatine levels, with decreases in glycine, o-phosphocholine, threonine, and valine. These metabolic findings were accompanied by a greater degradation index of the ECM in senescent H-Ras cells than in control H-Ras cells. MRS studies with the cell perfusion system revealed elevated creatine levels in senescent cells on Day 4, confirming the 1H-NMR results. These senescence-associated changes in metabolism and ECM degradation strongly impact growth and redox metabolism and reveal potential MRS signals for detecting senescent cancer cells in vivo.

SHG fiberscopy assessment of collagen morphology and its potential for breast cancer optical histology.

OBJECTIVE: this study is to investigate the feasibility of our recently developed nonlinear fiberscope for label-free in situ breast tumor detection and lymph node status assessment based on second harmonic generation (SHG) imaging of fibrillar collagen matrix with histological details. The long-term goal is to improve the current biopsy-based cancer paradigm with reduced sampling errors. METHODS: in this pilot study we undertook retrospective SHG imaging study of ex vivo invasive ductal carcinoma human biopsy tissue samples, and carried out quantitative image analysis to search for collagen structural signatures that are associated with the malignance of breast cancer. RESULTS: SHG fiberscopy image-based quantitative assessment of collagen fiber morphology reveals that: 1) cancerous tissues contain generally less extracellular collagen fibers compared with tumor-adjacent normal tissues, and 2) collagen fibers in lymph node positive biopsies are more aligned than lymph node negative counterparts. CONCLUSION/SIGNIFICANCE: the results demonstrate the promising potential of our SHG fiberscope for in situ breast tumor detection and lymph node involvement assessment and for offering real-time guidance during ongoing tissue biopsy.

Acidosis-mediated increase in IFN-γ-induced PD-L1 expression on cancer cells as an immune escape mechanism in solid tumors.

Immune checkpoint inhibitors have revolutionized cancer therapy, yet the efficacy of these treatments is often limited by the heterogeneous and hypoxic tumor microenvironment (TME) of solid tumors. In the TME, programmed death-ligand 1 (PD-L1) expression on cancer cells is mainly regulated by Interferon-gamma (IFN-γ), which induces T cell exhaustion and enables tumor immune evasion. In this study, we demonstrate that acidosis, a common characteristic of solid tumors, significantly increases IFN-γ-induced PD-L1 expression on aggressive cancer cells, thus promoting immune escape. Using preclinical models, we found that acidosis enhances the genomic expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and the translation of STAT1 mRNA by eukaryotic initiation factor 4F (elF4F), resulting in an increased PD-L1 expression. We observed this effect in murine and human anti-PD-L1-responsive tumor cell lines, but not in anti-PD-L1-nonresponsive tumor cell lines. In vivo studies fully validated our in vitro findings and revealed that neutralizing the acidic extracellular tumor pH by sodium bicarbonate treatment suppresses IFN-γ-induced PD-L1 expression and promotes immune cell infiltration in responsive tumors and thus reduces tumor growth. However, this effect was not observed in anti-PD-L1-nonresponsive tumors. In vivo experiments in tumor-bearing IFN-γ-/- mice validated the dependency on immune cell-derived IFN-γ for acidosis-mediated cancer cell PD-L1 induction and tumor immune escape. Thus, acidosis and IFN-γ-induced elevation of PD-L1 expression on cancer cells represent a previously unknown immune escape mechanism that may serve as a novel biomarker for anti-PD-L1/PD-1 treatment response. These findings have important implications for the development of new strategies to enhance the efficacy of immunotherapy in cancer patients.

PET/MRI and Bioluminescent Imaging Identify Hypoxia as a Cause of Programmed Cell Death Ligand 1 Image Heterogeneity.

Purpose To examine the association between hypoxia and programmed cell death ligand 1 (PD-L1) expression using bioluminescence imaging (BLI) and PET/MRI in a syngeneic mouse model of triple-negative breast cancer (TNBC). Materials and Methods PET/MRI and optical imaging were used to determine the role of hypoxia in altering PD-L1 expression using a syngeneic TNBC model engineered to express luciferase under hypoxia. Results Imaging showed a close spatial association between areas of hypoxia and increased PD-L1 expression in the syngeneic murine (4T1) tumor model. Mouse and human TNBC cells exposed to hypoxia exhibited a significant increase in PD-L1 expression, consistent with the in vivo imaging data. The role of hypoxia in increasing PD-L1 expression was further confirmed by using The Cancer Genome Atlas analyses of different human TNBCs. Conclusion These results have identified the potential role of hypoxia in contributing to PD-L1 heterogeneity in tumors by increasing cancer cell PD-L1 expression. Keywords: Hypoxia, PD-L1, Triple-Negative Breast Cancer, PET/MRI, Bioluminescence Imaging Supplemental material is available for this article. © RSNA, 2023.

Reprogramming of VEGF-mediated extracellular matrix changes through autocrine signaling.

Vascular endothelial growth factor (VEGF) plays key roles in angiogenesis, vasculogenesis, and wound healing. In cancers, including triple negative breast cancer (TNBC), VEGF has been associated with increased invasion and metastasis, processes that require cancer cells to traverse through the extracellular matrix (ECM) and establish angiogenesis at distant sites. To further understand the role of VEGF in modifying the ECM, we characterized VEGF-mediated changes in the ECM of tumors derived from TNBC MDA-MB-231 cells engineered to overexpress VEGF. We established that increased VEGF expression by these cells resulted in tumors with reduced collagen 1 (Col1) fibers, fibronectin, and hyaluronan. Molecular characterization of tumors identified an increase of MMP1, uPAR, and LOX, and a decrease of MMP2, and ADAMTS1. α-SMA, a marker of cancer associated fibroblasts (CAFs), increased, and FAP-α, a marker of a subset of CAFs associated with immune suppression, decreased with VEGF overexpression. Analysis of human data from The Cancer Genome Atlas Program confirmed mRNA differences for several molecules when comparing TNBC with high and low VEGF expression. We additionally characterized enzymatic changes induced by VEGF overexpression in three different cancer cell lines that clearly identified autocrine-mediated changes, specifically uPAR, in these enzymes. Unlike the increase of Col1 fibers and fibronectin mediated by VEGF during wound healing, in the TNBC model, VEGF significantly reduced key protein components of the ECM. These results further expand our understanding of the role of VEGF in cancer progression and identify potential ECM-related targets to disrupt this progression.

Functional roles of FAP-α in metabolism, migration and invasion of human cancer cells.

Fibroblast activation protein-α (FAP-α) is a transmembrane serine protease that is attracting significant interest as it is expressed by a subgroup of cancer-associated fibroblasts that play a role in immune suppression and cancer metastasis. FAP-α is also expressed by some cancer cells, such as melanoma, colorectal and breast cancer cells. Triple negative breast cancer (TNBC) is an aggressive cancer that urgently requires identification of novel targets for therapy. To expand our understanding of the functional roles of FAP-α in TNBC we engineered a human TNBC cell line, MDA-MB-231, to stably overexpress FAP-α and characterized changes in metabolism by 1H magnetic resonance spectroscopy, cell proliferation, migration characterized by wound healing, and invasion. FAP-α overexpression resulted in significant alterations in myoinositol, choline metabolites, creatine, and taurine, as well as a significant increase of migration and invasion, although proliferation remained unaltered. The increase of migration and invasion are consistent with the known activities of FAP-α as an exopeptidase and endopeptidase/gelatinase/collagenase in tissue remodeling and repair, and in cell migration. We additionally determined the effects of FAP-α overexpression on the human fibrosarcoma HT1080 cell line that showed increased migration, accompanied by limited changes in metabolism that identified the dependency of the metabolic changes on cell type. These metabolic data identify a previously unknown role of FAP-α in modifying cancer cell metabolism in the TNBC cell line studied here that may provide new insights into its functional roles in cancer progression.

Evaluating near-infrared photoimmunotherapy for targeting fibroblast activation protein-α expressing cells in vitro and in vivo.

Photoimmunotherapy (PIT), carried out using an Ab conjugated to the near infrared dye IRDye700DX, is achieving significant success in target-specific elimination of cells. Fibroblast activation protein alpha (FAP-α) is an important target in cancer because of its expression by cancer-associated fibroblasts (CAFs) as well as by some cancer cells. Cancer-associated fibroblasts that express FAP-α have protumorigenic and immune suppressive functions. Using immunohistochemistry of human breast cancer tissue microarrays, we identified an increase of FAP-α+  CAFs in invasive breast cancer tissue compared to adjacent normal tissue. We found FAP-α expression increased in fibroblasts cocultured with cancer cells. In proof-of-principle studies, we engineered human FAP-α overexpressing MDA-MB-231 and HT-1080 cancer cells and murine FAP-α overexpressing NIH-3T3 fibroblasts to evaluate several anti-FAP-α Abs and selected AF3715 based on its high binding affinity with both human and mouse FAP-α. After conjugation of AF3715 with the phthalocyanine dye IR700, the resultant Ab conjugate, FAP-α-IR700, was evaluated in cells and tumors for its specificity and effectiveness in eliminating FAP-α expressing cell populations with PIT. Fibroblast activation protein-α-IR700-PIT resulted in effective FAP-α-specific cell killing in the engineered cancer cells and in two patient-derived CAFs in a dose-dependent manner. Following an intravenous injection, FAP-α-IR700 retention was three-fold higher than IgG-IR700 in FAP-α overexpressing tumors, and two-fold higher compared to WT tumors. Fibroblast activation protein-α-IR700-PIT resulted in significant growth inhibition of tumors derived from FAP-α overexpressing human cancer cells. A reduction of endogenous FAP-α+ murine CAFs was identified at 7 days after FAP-α-IR700-PIT. Fibroblast activation protein-α-targeted near infrared PIT presents a promising strategy to eliminate FAP-α+ CAFs.

Cancer insights from magnetic resonance spectroscopy of cells and excised tumors.

Multinuclear ex vivo magnetic resonance spectroscopy (MRS) of cancer cells, xenografts, human cancer tissue, and biofluids is a rapidly expanding field that is providing unique insights into cancer. Starting from the 1970s, the field has continued to evolve as a stand-alone technology or as a complement to in vivo MRS to characterize the metabolome of cancer cells, cancer-associated stromal cells, immune cells, tumors, biofluids and, more recently, changes in the metabolome of organs induced by cancers. Here, we review some of the insights into cancer obtained with ex vivo MRS and provide a perspective of future directions. Ex vivo MRS of cells and tumors provides opportunities to understand the role of metabolism in cancer immune surveillance and immunotherapy. With advances in computational capabilities, the integration of artificial intelligence to identify differences in multinuclear spectral patterns, especially in easily accessible biofluids, is providing exciting advances in detection and monitoring response to treatment. Metabolotheranostics to target cancers and to normalize metabolic changes in organs induced by cancers to prevent cancer-induced morbidity are other areas of future development.

Twist alters the breast tumor microenvironment via choline kinase to facilitate an aggressive phenotype.

Twist (TWIST1) is a gene required for cell fate specification in embryos and its expression in mammary epithelium can initiate tumorigenesis through the epithelial-mesenchymal transition. To identify downstream target genes of Twist in breast cancer, we performed microarray analysis on the transgenic breast cancer cell line, MCF-7/Twist. One of the targets identified was choline kinase whose upregulation resulted in increased cellular phosphocholine and total choline containing compounds-a characteristic observed in highly aggressive metastatic cancers. To study the interactions between Twist, choline kinase, and their effect on the microenvironment, we used 1H magnetic resonance spectroscopy and found significantly higher phosphocholine and total choline, as well as increased phosphocholine/glycerophosphocholine ratio in MCF-7/Twist cells. We also observed significant increases in extracellular glucose, lactate, and [H +] ion concentrations in the MCF-7/Twist cells. Magnetic resonance imaging of MCF-7/Twist orthotopic breast tumors showed a significant increase in vascular volume and permeability surface area product compared to control tumors. In addition, by reverse transcription-quantitative polymerase chain reaction, we discovered that Twist upregulated choline kinase expression in estrogen receptor negative breast cancer cell lines through FOXA1 downregulation. Moreover, using The Cancer Genome Atlas database, we observed a significant inverse relationship between FOXA1 and choline kinase expression and propose that it could act as a modulator of the Twist/choline kinase axis. The data presented indicate that Twist is a driver of choline kinase expression in breast cancer cells via FOXA1 resulting in the generation of an aggressive breast cancer phenotype.

PSMA-specific degradable dextran for multiplexed immunotargeted siRNA therapeutics against prostate cancer.

Small interfering RNA (siRNA) is ideal for gene silencing through a sequence-specific RNA interference process. The redundancy and complexity of molecular pathways in cancer create a need for multiplexed targeting that can be achieved with multiplexed siRNA delivery. Here, we delivered multiplexed siRNA with a PSMA-targeted biocompatible dextran nanocarrier to downregulate CD46 and PD-L1 in PSMA expressing prostate cancer cells. The selected gene targets, PD-L1 and CD46, play important roles in the escape of cancer cells from immune surveillance. PSMA, abundantly expressed by prostate cancer cells, allowed the prostate cancer-specific delivery of the nanocarrier. The nanocarrier was modified with acid cleavable acetal bonds for a rapid release of siRNA. Cell imaging and flow cytometry studies confirmed the PSMA-specific delivery of CD46 and PD-L1 siRNA to high PSMA expressing PC-3 PIP cells. Immunoblot, qRT-PCR and flow cytometry methods confirmed the downregulation of CD46 and PD-L1 following treatment with multiplexed siRNA.

Characterization of lipomatous tumors with high-resolution 1H MRS at 17.6T: Do benign lipomas, atypical lipomatous tumors and liposarcomas have a distinct metabolic signature?

Background: Distinguishing between some benign lipomas (BLs), atypical lipomatous tumors (ALTs), and dedifferentiated liposarcomas (DDLs) can be challenging due to overlapping magnetic resonance imaging characteristics, and poorly understood molecular mechanisms underlying the malignant transformation of liposarcomas. Purpose: To identify metabolic biomarkers of the lipomatous tumor spectrum by examining human tissue specimens using high-resolution 1H magnetic resonance spectroscopy (MRS). Materials and methods: In this prospective study, human tissue specimens were obtained from participants who underwent surgical resection for radiologically-indeterminate lipomatous tumors between November 2016 and May 2019. Tissue specimens were obtained from normal subcutaneous fat (n=9), BLs (n=10), ALTs (n=7) and DDLs (n=8). Extracts from specimens were examined with high-resolution MRS at 17.6T. Computational modeling of pattern recognition-based cluster analysis was utilized to identify significant differences in metabolic signatures between the lipomatous tumor types. Results: Significant differences between BLs and ALTs were observed for multiple metabolites, including leucine, valine, branched chain amino acids, alanine, acetate, glutamine, and formate. DDLs were distinguished from ALTs by increased glucose and lactate, and increased phosphatidylcholine. Multivariate principal component analysis showed clear clustering identifying distinct metabolic signatures of the tissue types. Conclusion: Metabolic signatures identified in 1H MR spectra of lipomatous tumors provide new insights into malignant progression and metabolic targeting. The metabolic patterns identified provide the foundation of developing noninvasive MRS or PET imaging biomarkers to distinguish between BLs, ALTs, and DDLs.

Phototheranostics of Splenic Myeloid-Derived Suppressor Cells and Its Impact on Spleen Metabolism in Tumor-Bearing Mice.

(1) Background: MDSCs play an active role in the immune surveillance escape of cancer cells. Because MDSCs in mice are CD11b+Gr1+, near-infrared photoimmunotherapy (NIR-PIT) using the NIR dye IR700 conjugated to an MDSC-binding antibody provides an opportunity for targeted elimination of MDSCs. (2) Methods: The efficacy of Gr1-IR700-mediated NIR-PIT was evaluated in vitro using magnetically separated CD11b+Gr1+ MDSCs from spleens of 4T1-luc tumor-bearing (TB) mice. For in vivo evaluation, spleens of Gr1-IR700-injected 4T1-luc TB mice were irradiated with NIR light, and splenocyte viability was determined using CCK-8 assays. Metabolic profiling of NIR-PIT-irradiated spleens was performed using 1H MRS. (3) Results: Flow cytometric analysis confirmed a ten-fold increase in splenic MDSCs in 4T1-luc TB mice. Gr1-IR700-mediated NIR-PIT eliminated tumor-induced splenic MDSCs in culture. Ex vivo fluorescence imaging revealed an 8- and 9-fold increase in mean fluorescence intensity (MFI) in the spleen and lungs of Gr1-IR700-injected compared to IgG-IR700-injected TB mice. Splenocytes from Gr1-IR700-injected TB mice exposed in vivo to NIR-PIT demonstrated significantly lower viability compared to no light exposure or untreated control groups. Significant metabolic changes were observed in spleens following NIR-PIT. (4) Conclusions: Our data confirm the ability of NIR-PIT to eliminate splenic MDSCs, identifying its potential to eliminate MDSCs in tumors to reduce immune suppression. The metabolic changes observed may identify potential biomarkers of splenic MDSC depletion as well as potential metabolic targets of MDSCs.

VEGF Overexpression Significantly Increases Nanoparticle-Mediated siRNA Delivery and Target-Gene Downregulation.

The availability of nanoparticles (NPs) to deliver small interfering RNA (siRNA) has significantly expanded the specificity and range of 'druggable' targets for precision medicine in cancer. This is especially important for cancers such as triple negative breast cancer (TNBC) for which there are no targeted treatments. Our purpose here was to understand the role of tumor vasculature and vascular endothelial growth factor (VEGF) overexpression in a TNBC xenograft in improving the delivery and function of siRNA NPs using in vivo as well as ex vivo imaging. We used triple negative MDA-MB-231 human breast cancer xenografts derived from cells engineered to overexpress VEGF to understand the role of VEGF and vascularization in NP delivery and function. We used polyethylene glycol (PEG) conjugated polyethylenimine (PEI) NPs to deliver siRNA that downregulates choline kinase alpha (Chkα), an enzyme that is associated with malignant transformation and tumor progression. Because Chkα converts choline to phosphocholine, effective delivery of Chkα siRNA NPs resulted in functional changes of a significant decrease in phosphocholine and total choline that was detected with 1H magnetic resonance spectroscopy (MRS). We observed a significant increase in NP delivery and a significant decrease in Chkα and phosphocholine in VEGF overexpressing xenografts. Our results demonstrated the importance of tumor vascularization in achieving effective siRNA delivery and downregulation of the target gene Chkα and its function.

Two diverse carriers are better than one: A case study in α-particle therapy for prostate specific membrane antigen-expressing prostate cancers.

Partial and/or heterogeneous irradiation of established (i.e., large, vascularized) tumors by α-particles that exhibit only a 4-5 cell-diameter range in tissue, limits the therapeutic effect, since regions not being hit by the high energy α-particles are likely not to be killed. This study aims to mechanistically understand a delivery strategy to uniformly distribute α-particles within established solid tumors by simultaneously delivering the same α-particle emitter by two diverse carriers, each killing a different region of the tumor: (1) the cancer-agnostic, but also tumor-responsive, liposomes engineered to best irradiate tumor regions far from the vasculature, and (2) a separately administered, antibody, targeting any cancer-cell's surface marker, to best irradiate the tumor perivascular regions. We demonstrate that on a prostate specific membrane antigen (PSMA)-expressing prostate cancer xenograft mouse model, for the same total injected radioactivity of the α-particle emitter Actinium-225, any radioactivity split ratio between the two carriers resulted in better tumor growth inhibition compared to the tumor inhibition when the total radioactivity was delivered by any of the two carriers alone. This finding was due to more uniform tumor irradiation for the same total injected radioactivity. The killing efficacy was improved even though the tumor-absorbed dose delivered by the combined carriers was lower than the tumor-absorbed dose delivered by the antibody alone. Studies on spheroids with different receptor-expression, used as surrogates of the tumors' avascular regions, demonstrated that our delivery strategy is valid even for as low as 1+ (ImmunoHistoChemistry score) PSMA-levels. The findings presented herein may hold clinical promise for those established tumors not being effectively eradicated by current α-particle radiotherapies.

PD-L1 near Infrared Photoimmunotherapy of Ovarian Cancer Model.

(1) Background: Despite advances in surgical approaches and drug development, ovarian cancer is still a leading cause of death from gynecological malignancies. Patients diagnosed with late-stage disease are treated with aggressive surgical resection and chemotherapy, but recurrence with resistant disease is often observed following treatment. There is a critical need for effective therapy for late-stage ovarian cancer. Photoimmunotherapy (PIT), using an antibody conjugated to a near infrared (NIR) dye, constitutes an effective theranostic strategy to detect and selectively eliminate targeted cell populations. (2) Methods: Here, we are targeting program death ligand 1 (PD-L1) using NIR-PIT in a syngeneic mouse model of ovarian cancer. PD-L1 PIT-mediated cytotoxicity was quantified in RAW264.7 macrophages and ID8-Defb29-VEGF cells in culture, and in vivo with orthotopic ID8-Defb29-VEGF tumors. (3) Results: Treatment efficacy was observed both in vitro and in vivo. (4) Conclusions: Our data highlight the need for further investigations to assess the potential of using NIR-PIT for ovarian cancer therapy to improve the treatment outcome of ovarian cancer.

Deep learning-based classification of preclinical breast cancer tumor models using chemical exchange saturation transfer magnetic resonance imaging.

Chemical exchange saturation transfer (CEST) magnetic resonance imaging has shown promise for classifying tumors based on their aggressiveness, but CEST contrast is complicated by multiple signal sources and thus prolonged acquisition times are often required to extract the signal of interest. We investigated whether deep learning could help identify pertinent Z-spectral features for distinguishing tumor aggressiveness as well as the possibility of acquiring only the pertinent spectral regions for more efficient CEST acquisition. Human breast cancer cells, MDA-MB-231 and MCF-7, were used to establish bi-lateral tumor xenografts in mice to represent higher and lower aggressive tumors, respectively. A convolutional neural network (CNN)-based classification model, trained on simulated data, utilized Z-spectral features as input to predict labels of different tissue types, including MDA-MB-231, MCF-7, and muscle tissue. Saliency maps reported the influence of Z-spectral regions on classifying tissue types. The model was robust to noise with an accuracy of more than 91.5% for low and moderate noise levels in simulated testing data (SD of noise less than 2.0%). For in vivo CEST data acquired with a saturation pulse amplitude of 2.0 µT, the model had a superior ability to delineate tissue types compared with Lorentzian difference (LD) and magnetization transfer ratio asymmetry (MTRasym ) analysis, classifying tissues to the correct types with a mean accuracy of 85.7%, sensitivity of 81.1%, and specificity of 94.0%. The model's performance did not improve substantially when using data acquired at multiple saturation pulse amplitudes or when adding LD or MTRasym spectral features, and did not change when using saliency map-based partial or downsampled Z-spectra. This study demonstrates the potential of CNN-based classification to distinguish between different tumor types and muscle tissue, and speed up CEST acquisition protocols.

Combination of Carriers with Complementary Intratumoral Microdistributions of Delivered α-Particles May Realize the Promise for 225Ac in Large, Solid Tumors.

α-particle radiotherapy has already been shown to be impervious to most resistance mechanisms. However, in established (i.e., large, vascularized) soft-tissue lesions, the diffusion-limited penetration depths of radiolabeled antibodies or nanocarriers (≤50-80 µm) combined with the short range of α-particles (4-5 cell diameters) may result in only partial tumor irradiation, potentially limiting treatment efficacy. To address this challenge, we combined carriers with complementary intratumoral microdistributions of the delivered α-particles. We used the α-particle generator 225Ac, and we combined a tumor-responsive liposome (which, on tumor uptake, releases into the interstitium a highly diffusing form of its radioactive payload [225Ac-DOTA], potentially penetrating the deeper parts of tumors where antibodies do not reach) with a separately administered, less-penetrating radiolabeled antibody (irradiating the tumor perivascular regions where liposome contents clear too quickly). Methods: In a murine model with orthotopic human epidermal growth factor receptor 2-positive BT474 breast cancer xenografts, the biodistributions of each carrier were evaluated, and the control of tumor growth was monitored after administration of the same total radioactivity of 225Ac delivered by the 225Ac-DOTA-encapsulating liposomes, by the 225Ac-DOTA-SCN--labeled trastuzumab, and by both carriers at equally split radioactivities. Results: Tumor growth was significantly more inhibited when the same total injected radioactivity was divided between the 2 separate carriers than when delivered by either of the carriers alone. The combined carriers enabled more uniform intratumoral microdistributions of α-particles, at a tumor dose that was lower than the dose delivered by the antibody alone. Conclusion: This strategy demonstrates that more uniform microdistributions of the delivered α-particles within established solid tumors improve efficacy even at lower tumor doses. Augmentation of antibody-targeted α-particle therapies with tumor-responsive liposomes may address partial tumor irradiation, improving therapeutic effects.