Tumor-associated vascular dysfunction is a major barrier limiting the efficacy of cancer therapies by impairing drug delivery, restricting immune-cell infiltration, and promoting hypoxia within the tumor microenvironment (TME). While anti-angiogenic therapies aim to suppress abnormal vessel formation, excessive vascular inhibition may further worsen hypoxia and therapeutic resistance. Here, we investigated whether restoration of endothelial integrity using Poloxamer 188 (P188), a membrane-stabilizing triblock copolymer, could improve vascular function and enhance therapeutic efficacy in triple-negative breast cancer (TNBC). In vitro, P188 prevented and reversed endothelial leakiness, restoring endothelial barrier integrity and enhancing tight junction formation, as demonstrated by increased Zonula occludens-1 (ZO-1) expression, a tight-junction protein, and sealed endothelial morphology. In vivo, fluorescently labeled P188 accumulated in TNBC tumors and significantly enhanced Dextran delivery, indicating improved vascular function and tumor perfusion. Although P188 showed no direct cytotoxic effect on TNBC cells and did not enhance Doxorubicin activity in vitro, combination treatment significantly suppressed tumor progression in vivo, while simultaneously reducing treatment-associated weight loss. Importantly, P188 also enhanced antitumor immunity. While P188 did not directly activate T cells or increase their cytotoxicity in vitro, it significantly increased T-cell infiltration into tumors in vivo and enhanced the therapeutic efficacy of adoptively transferred human normal peripheral blood mononuclear cell (PBMCs). Collectively, these findings demonstrate that restoration of tumor vascular integrity using P188 improves both chemotherapy and immunotherapy efficacy by enhancing therapeutic delivery and immune-cell infiltration. This study highlights vascular restoration as a promising therapeutic strategy for overcoming TME-mediated resistance in solid tumors.

Multiple myeloma (MM) is the cancer of plasma cells within the bone marrow and remains incurable. Tumor-associated macrophages (TAMs) within the tumor microenvironment often display a pro-tumor phenotype and correlate with tumor proliferation, survival, and therapy resistance. IL-10 is a key immunosuppressive cytokine that leads to recruitment and development of TAMs. In this study, we investigated the role of IL-10 in MM TAM development as well as the therapeutic application of IL-10/IL-10R/STAT3 signaling inhibition. We demonstrated that IL-10 is overexpressed in MM BM and mediates M2-like polarization of TAMs in patient BM, 3D co-cultures in vitro, and mouse models. In turn, TAMs promote MM proliferation and drug resistance, both in vitro and in vivo. Moreover, inhibition of IL-10/IL-10R/STAT3 axis using a blocking IL-10R monoclonal antibody and STAT3 protein degrader/PROTAC prevented M2 polarization of TAMs and the consequent TAM-induced proliferation of MM, and re-sensitized MM to therapy, in vitro and in vivo. Therefore, our findings suggest that inhibition of IL-10/IL-10R/STAT3 axis is a novel therapeutic strategy with monotherapy efficacy and can be further combined with current anti-MM therapy, such as immunomodulatory drugs, to overcome drug resistance. Future investigation is warranted to evaluate the potential of such therapy in MM patients.

Supplementary Table 3 from Identification of Copy Number Abnormalities and Inactivating Mutations in Two Negative Regulators of Nuclear Factor-κB Signaling Pathways in Waldenström's Macroglobulinemia

Supplementary Data from Src Tyrosine Kinase Regulates Adhesion and Chemotaxis in Waldenstrom Macroglobulinemia

Supplementary Table 7 from Identification of Copy Number Abnormalities and Inactivating Mutations in Two Negative Regulators of Nuclear Factor-κB Signaling Pathways in Waldenström's Macroglobulinemia

Supplementary Table 2 from Identification of Copy Number Abnormalities and Inactivating Mutations in Two Negative Regulators of Nuclear Factor-κB Signaling Pathways in Waldenström's Macroglobulinemia

Supplementary Table 5 from Identification of Copy Number Abnormalities and Inactivating Mutations in Two Negative Regulators of Nuclear Factor-κB Signaling Pathways in Waldenström's Macroglobulinemia

Supplementary Table 4 from Identification of Copy Number Abnormalities and Inactivating Mutations in Two Negative Regulators of Nuclear Factor-κB Signaling Pathways in Waldenström's Macroglobulinemia

Supplementary Table 6 from Identification of Copy Number Abnormalities and Inactivating Mutations in Two Negative Regulators of Nuclear Factor-κB Signaling Pathways in Waldenström's Macroglobulinemia

PDF file - 32K, Everolimus targets mTOR-down stream targets in WM patients. Bone marrow specimens have been stained for phosphor(p)-S6R and p-4EBP1. Signal quantification for each target has been obtained from 4 different areas, on matched pre- and post-everolimus treatment of 4 patients presenting with partial response; and on matched pre- and post-everolimus treatment of 3 patients presenting with stable disease. Number of positive cells has been obtained on 4 different fields of the bone marrow biopsy, and average and standard deviation provided. P indicates P values; N.S.: not significant.

Supplementary Figures S1-S3.

Supplementary Figure 1 from Identification of Copy Number Abnormalities and Inactivating Mutations in Two Negative Regulators of Nuclear Factor-κB Signaling Pathways in Waldenström's Macroglobulinemia

Supplementary Figure Legends 1-2 from Identification of Copy Number Abnormalities and Inactivating Mutations in Two Negative Regulators of Nuclear Factor-κB Signaling Pathways in Waldenström's Macroglobulinemia

Abstract Introduction: Multiple myeloma (MM) is the cancer of plasma cells within the bone marrow (BM) and represents the second most common hematologic malignancy. Although therapeutic options have broadened over the years, the disease is challenged by frequent relapses. Relapsed/refractory MM (RRMM) often becomes non-responsive to previous lines of treatment and has significantly poorer survival outcome. Physicians are faced with a difficult task to choose a right treatment regimen. Thus, a precision medicine tool that predicts the clinical response of individual patients to therapy is greatly desired. We have previously developed a novel 3D tissue-engineered BM (3DTEBM) culture model, which is patient derived, closely recapitulates the pathophysiological conditions in the BM, and allows ex vivo proliferation of primary cells of various hematologic malignancies. In this study, we conducted a retrospective study that tests the ability of the ex vivo 3DTEBM platform to predict the clinical response in individual MM patients, to help decision-making process for RRMM. We hypothesized that the 3DTEBM will be able to predict clinical responses of RRMM patients. Methods: We first performed a literature search to examine the clinical efficacious concentrations (Css) for 10 MM drugs. We then experimentally determined the in vitro efficacious concentrations (IC50) of these drugs in MM cell lines, in both 2D and 3DTEBM cultures. The IC50 values were then correlated with their respective clinical Css values to evaluate how well each culture system reproduce drug efficacy. For the retrospective trial, we used viably frozen whole BM samples from 19 RRMM patients with known clinical responsiveness to the regimen they received. 3DTEBM cultures were developed for each patient with BM biopsies obtained prior to the start of clinical regimen. Cultures were treated ex vivo with the same treatment regimen each patient received clinically, at increasing concentrations (0X, 3X and 10X of Css of individual drugs). After 4 days, cultures were digested and cells were retrieved for flow cytometry analysis. Primary cells were stained Leukocyte-/CD38+ and counted against counting beads. Survival was determined as % of untreated control, and ex vivo responsiveness was analyzed by ANOVA. Finally, the clinical team correlated the ex vivo response with the clinical response for each patient. Results: To demonstrate this discrepancy between drug efficacy in laboratory settings and clinical outcomes, we compared the in vitro IC50 to the clinical Css of 10 drugs used for the treatment of MM. We found that there was no correlation between the IC50 in classic 2D culture systems and the clinical Css (R 2=0.019) (Figure 1A). In contrast, the IC50 in the 3DTEBM directly correlated with the clinical Css (R 2=0.993) (Figure 1B). We then conducted a retrospective clinical trial to determine if the 3DTEBM platform is able to predict each patient's clinical response by recreating the same treatment regimen ex vivo (Figure 1C). The 3DTEBM was able to predict the response in 89% of the MM patient cohort across multiple treatment regimens, with no false positives (Figure 1D). Conclusions: Our retrospective clinical trial demonstrated that the 3DTEBM technology is a feasible platform for predicting therapeutic responses in MM with a high predictive accuracy within a clinically actionable time frame. Such platforms can provide precise clinical insight about the efficacy of different treatment plans and assist physicians to propose the best choice of therapy for their individual patients. Future prospective studies are needed to validate these significant findings by testing prospective prediction ability of 3DTEBM to improve therapy response in hematologic malignancies. Figure 1 Figure 1. Disclosures De La Puente: Cellatrix LLC: Other: Co-founder. Azab: Cellatrix LLC: Current Employment. Vij: BMS: Research Funding; Takeda: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; BMS: Honoraria; GSK: Honoraria; Oncopeptides: Honoraria; Karyopharm: Honoraria; CareDx: Honoraria; Legend: Honoraria; Biegene: Honoraria; Adaptive: Honoraria; Harpoon: Honoraria. Azab: Cellatrix, LLC: Current Employment, Current holder of individual stocks in a privately-held company.

Cancer patients undergo detrimental toxicities and ineffective treatments especially in the relapsed setting, due to failed treatment attempts. The development of a tool that predicts the clinical response of individual patients to therapy is greatly desired. We have developed a novel patient-derived 3D tissue engineered bone marrow (3DTEBM) technology that closely recapitulate the pathophysiological conditions in the bone marrow and allows ex vivo proliferation of tumor cells of hematologic malignancies. In this study, we used the 3DTEBM to predict the clinical response of individual multiple myeloma (MM) patients to different therapeutic regimens. We found that while no correlation was observed between in vitro efficacy in classic 2D culture systems of drugs used for MM with their clinical efficacious concentration, the efficacious concentration in the 3DTEBM were directly correlated. Furthermore, the 3DTEBM model retrospectively predicted the clinical response to different treatment regimens in 89% of the MM patient cohort. These results demonstrated that the 3DTEBM is a feasible platform which can predict MM clinical responses with high accuracy and within a clinically actionable time frame. Utilization of this technology to predict drug efficacy and the likelihood of treatment failure could significantly improve patient care and treatment in many ways, particularly in the relapsed and refractory setting. Future studies are needed to validate the 3DTEBM model as a tool for predicting clinical efficacy.

Background CD138 has been the gold-standard surface marker to detect multiple myeloma (MM) cells for decades; however, drug resistant minimal-residual disease (MRD) and circulating tumor cells (CTCs) were shown to have lower expression of this marker. We previously published that residual MM cells following treatment in vivo were hypoxic and the combination of hypoxia and chemotherapy such as bortezomib downregulated CD138 expression, thereby making this marker unsuitable for MM detection. Needless to say, accurate number of MM cells is critical in diagnosis, autologous transplantation, MRD and drug efficiency assessment. Moreover, CTCs are considered an unfavorable prognostic factor and indicate an aggressive form of the disease, and therefore detecting CTCs can be used as a powerful prognostic tool for MM. Methods We used an alternative biomarker-set using flow cytometry defining MM cells as any cell that expresses CD38 but excluding T cell (CD3), B cell (CD19), NK cell (CD16), monocyte/macrophage (CD14), neutrophil/eosinophil (CD16), and basophil/dendritic cell (CD123). We demonstrated previously that this approach it widely available due to accessibility of flow cytometry, inexpensive, and works independently of hypoxic-, CD138 expression- and treatment status. We analyzed primary patient samples (n=50) with complete response or very good partial response for MRD and CTCs and correlated the numbers of detected MM with patients’ time-to-progression (TTP) obtained from a clinical data base. Results We found that the alternative biomarker-set identifies MM cells more precisely and at higher numbers than CD138 marker by flow or histology. Moreover, we found a correlation between the number of MM cells detected and TTP in these patients: the amount of MM cells detected by the new method ranged between 0.5-7.3%, and patients who progressed sooner than two years had 2.5-fold higher percentage of MM cells compared to patients who relapsed later than 2 years. Similarly, patients who relapsed sooner than 3 years had 4-fold higher number of MM cells than patients who relapsed later than 3 years. We further found that, among all patients who had more than 2% of MM cells detected by the new method had a mean TPP of about 20 months, while patients who had less than 2% had a mean TPP of about 38 months. Testing the prevalence of CTCs in MM patients with progressive disease using CD138 or the new method, demonstrated that CD138 detected minimal amounts of MM cells in all patients (less than 0.1%), while the new method detected a range between 0.1 - 1.8% of MM cells in the peripheral blood. Conclusion These results suggest that the alternative biomarker-set detected MM cells which correlated with relapse in MM patients. Therefore, the amount of residual cells in the bone marrow and circulating myeloma cells can be used as a prognostic marker in MM patients. Further examination to characterize this population and its role in MM relapse is warranted. CD138 has been the gold-standard surface marker to detect multiple myeloma (MM) cells for decades; however, drug resistant minimal-residual disease (MRD) and circulating tumor cells (CTCs) were shown to have lower expression of this marker. We previously published that residual MM cells following treatment in vivo were hypoxic and the combination of hypoxia and chemotherapy such as bortezomib downregulated CD138 expression, thereby making this marker unsuitable for MM detection. Needless to say, accurate number of MM cells is critical in diagnosis, autologous transplantation, MRD and drug efficiency assessment. Moreover, CTCs are considered an unfavorable prognostic factor and indicate an aggressive form of the disease, and therefore detecting CTCs can be used as a powerful prognostic tool for MM. We used an alternative biomarker-set using flow cytometry defining MM cells as any cell that expresses CD38 but excluding T cell (CD3), B cell (CD19), NK cell (CD16), monocyte/macrophage (CD14), neutrophil/eosinophil (CD16), and basophil/dendritic cell (CD123). We demonstrated previously that this approach it widely available due to accessibility of flow cytometry, inexpensive, and works independently of hypoxic-, CD138 expression- and treatment status. We analyzed primary patient samples (n=50) with complete response or very good partial response for MRD and CTCs and correlated the numbers of detected MM with patients’ time-to-progression (TTP) obtained from a clinical data base. We found that the alternative biomarker-set identifies MM cells more precisely and at higher numbers than CD138 marker by flow or histology. Moreover, we found a correlation between the number of MM cells detected and TTP in these patients: the amount of MM cells detected by the new method ranged between 0.5-7.3%, and patients who progressed sooner than two years had 2.5-fold higher percentage of MM cells compared to patients who relapsed later than 2 years. Similarly, patients who relapsed sooner than 3 years had 4-fold higher number of MM cells than patients who relapsed later than 3 years. We further found that, among all patients who had more than 2% of MM cells detected by the new method had a mean TPP of about 20 months, while patients who had less than 2% had a mean TPP of about 38 months. Testing the prevalence of CTCs in MM patients with progressive disease using CD138 or the new method, demonstrated that CD138 detected minimal amounts of MM cells in all patients (less than 0.1%), while the new method detected a range between 0.1 - 1.8% of MM cells in the peripheral blood. These results suggest that the alternative biomarker-set detected MM cells which correlated with relapse in MM patients. Therefore, the amount of residual cells in the bone marrow and circulating myeloma cells can be used as a prognostic marker in MM patients. Further examination to characterize this population and its role in MM relapse is warranted.