Chemotherapy and the Extra-Tumor Immune Microenvironment: EXTRA-TIME.

SUMMARY: Understandably, conventional therapeutic strategies have focused on controlling primary tumors. We ask whether the cost of such strategies is actually an increased likelihood of metastatic relapse.

Induction of complementary immunogenic necroptosis and apoptosis cell death pathways inhibits cancer metastasis and relapse.

Interactions of light-sensitive drugs and materials with Cerenkov radiation-emitting radiopharmaceuticals generate cytotoxic reactive oxygen species (ROS) to inhibit localized and disseminated cancer progression, but the cell death mechanisms underlying this radionuclide stimulated dynamic therapy (RaST) remain elusive. Using ROS-regenerative nanophotosensitizers coated with a tumor-targeting transferrin-titanocene complex (TiO2-TC-Tf) and radiolabeled 2-fluorodeoxyglucose (18FDG), we found that adherent dying cells maintained metabolic activity with increased membrane permeabilization. Mechanistic assessment of these cells revealed that RaST activated the expression of RIPK-1 and RIPK-3, which mediate necroptosis cell death. Subsequent recruitment of the nuclear factors kappa B and the executioner mixed lineage kinase domain-like pseudo kinase (MLKL) triggered plasma membrane permeabilization and pore formation, respectively, followed by the release of cytokines and immunogenic damage-associated molecular patterns (DAMPs). In immune-deficient breast cancer models with adequate stroma and growth factors that recapitulate the human tumor microenvironment, RaST failed to inhibit tumor progression and the ensuing lung metastasis. A similar aggressive tumor model in immunocompetent mice responded to RaST, achieving a remarkable partial response (PR) and complete response (CR) with no evidence of lung metastasis, suggesting active immune system engagement. RaST recruited antitumor CD11b+, CD11c+, and CD8b+ effector immune cells after initiating dual immunogenic apoptosis and necroptosis cell death pathways in responding tumors in vivo. Over time, cancer cells upregulated the expression of negative immune regulating cytokine (TGF-ß) and soluble immune checkpoints (sICP) to challenge RaST effect in the CR mice. Using a signal-amplifying cancer-imaging agent, LS301, we identified latent minimal residual disseminated tumors in the lymph nodes (LNs) of the CR group. Despite increased protumor immunogens in the CR mice, RaST prevented cancer relapse and metastasis through dynamic redistribution of ROS-regenerative TiO2 from bones at the early treatment stage to the spleen and LNs, maintaining active immunity against cancer progression and migration. This study reveals the immune-mechanistic underpinnings of RaST-mediated antitumor immune response and highlights immunogenic reprogramming of tumors in response to RaST. Overcoming apoptosis resistance through complementary necroptosis activation paves the way for strategic drug combinations to improve cancer treatment.

HTLV-1 infected T cells cause bone loss via small extracellular vesicles.

Adult T cell leukemia (ATL), caused by infection with human T cell leukemia virus type 1 (HTLV-1), is often complicated by hypercalcemia and osteolytic lesions. Therefore, we studied the communication between patient-derived ATL cells (ATL-PDX) and HTLV-1 immortalized CD4+ T cell lines (HTLV/T) with osteoclasts and their effects on bone mass in mice. Intratibial inoculation of some HTLV/T lead to a profound local decrease in bone mass similar to marrow-replacing ATL-PDX, despite the fact that few HTLV/T cells persisted in the bone. To study the direct effect of HTLV/T and ATL-PDX on osteoclasts, supernatants were added to murine and human osteoclast precursors. ATL-PDX supernatants from hypercalcemic patients promoted formation of mature osteoclasts, while those from HTLV/T were variably stimulatory, but had largely consistent effects between human and murine cultures. Interestingly, this osteoclastic activity did not correlate with expression of osteoclastogenic cytokine RANKL, suggesting an alternative mechanism. HTLV/T and ATL-PDX produce small extracellular vesicles (sEV), known to facilitate HTLV-1 infection. We hypothesized that these sEV also mediate bone loss by targeting osteoclasts. We isolated sEV from both HTLV/T and ATL-PDX, and found they carried most of the activity found in supernatants. In contrast, sEV from uninfected activated T cells had little effect. Analysis of sEV (both active and inactive) by mass spectrometry and electron microscopy confirmed absence of RANKL and intact virus. Viral proteins Tax and Env were only present in sEV from the active, osteoclast-stimulatory group, along with increased representation of proteins involved in osteoclastogenesis and bone resorption. sEV injected over mouse calvaria in the presence of low dose RANKL caused more osteolysis than RANKL alone. Thus, HTLV-1 infection of T cells can cause release of sEV with strong osteolytic potential, providing a mechanism beyond RANKL production that modifies the bone microenvironment, even in the absence of overt leukemia.

BCAR4 Expression as a Predictive Biomarker for Endocrine Therapy Resistance in Breast Cancer.

BACKGROUND: Breast cancer, particularly the estrogen receptor positive (ER+) subtype, remains a leading cause of cancer-related death among women. Endocrine therapy is the most effective treatment for ER+ breast cancer; however, the development of resistance presents a significant challenge. This study explored the role of the breast cancer antiestrogen resistance 4 (BCAR4) gene as a potential driver of resistance and a pivotal biomarker in breast cancer. PATIENTS AND METHODS: The researchers undertook a comprehensive analysis of 1743 patients spanning 6 independent cohorts. They examined the association of BCAR4 expression with patient outcomes across all breast cancer types and the PAM50 molecular subtypes. The relationship between elevated BCAR4 expression and resistance to endocrine therapy including AIs, the prevailing standard-of-care for endocrine therapy, was also investigated. RESULTS: This meta-analysis corroborated the link between BCAR4 expression and adverse outcomes as well as resistance to endocrine therapy in breast cancer. Notably, BCAR4 expression is clinically significant in luminal A and B subtypes. Additionally, an association between BCAR4 expression and resistance to AI treatment was discerned. CONCLUSION: This study expands on previous findings by demonstrating that BCAR4 expression is associated with resistance to newer therapies. The identification of patients with intrinsic resistance to hormone therapy is crucial to avoid ineffective treatment strategies. These findings contribute to our understanding of endocrine therapy resistance in breast cancer and could potentially guide the development of more effective treatment strategies.

Resistance to abemaciclib is associated with increased metastatic potential and lysosomal protein deregulation in breast cancer cells.

Cyclin dependent kinase 4 and 6 inhibitors such as abemaciclib are routinely used to treat metastatic estrogen receptor positive (ER+) breast cancer. However, adaptive mechanisms inhibit their effectiveness and allow for disease progression. Using ER+ breast cancer cell models, we show that acquired resistance to abemaciclib is accompanied by increase in metastatic potential. Mass spectrometry-based proteomics from abemaciclib sensitive and resistant cells showed that lysosomal proteins including CTSD (cathepsin D), cathepsin A and CD68 were significantly increased in resistant cells. Combination of abemaciclib and a lysosomal destabilizer, such as hydroxychloroquine (HCQ) or bafilomycin A1, resensitized resistant cells to abemaciclib. Also, combination of abemaciclib and HCQ decreased migration and invasive potential and increased lysosomal membrane permeability in resistant cells. Prosurvival B cell lymphoma 2 (BCL2) protein levels were elevated in resistant cells, and a triple treatment with abemaciclib, HCQ, and BCL2 inhibitor, venetoclax, significantly inhibited cell growth compared to treatment with abemaciclib and HCQ. Furthermore, resistant cells showed increased levels of Transcription Factor EB (TFEB), a master regulator of lysosomal-autophagy genes, and siRNA mediated knockdown of TFEB decreased invasion in resistant cells. TFEB was found to be mutated in a subset of invasive human breast cancer samples, and overall survival analysis in ER+, lymph node-positive breast cancer showed that increased TFEB expression correlated with decreased survival. Collectively, we show that acquired resistance to abemaciclib leads to increased metastatic potential and increased levels of protumorigenic lysosomal proteins. Therefore, the lysosomal pathway could be a therapeutic target in advanced ER+ breast cancer.

Transferrin receptor in primary and metastatic breast cancer: Evaluation of expression and experimental modulation to improve molecular targeting.

BACKGROUND: Conjugation of transferrin (Tf) to imaging or nanotherapeutic agents is a promising strategy to target breast cancer. Since the efficacy of these biomaterials often depends on the overexpression of the targeted receptor, we set out to survey expression of transferrin receptor (TfR) in primary and metastatic breast cancer samples, including metastases and relapse, and investigate its modulation in experimental models. METHODS: Gene expression was investigated by datamining in twelve publicly-available datasets. Dedicated Tissue microarrays (TMAs) were generated to evaluate matched primary and bone metastases as well as and pre and post chemotherapy tumors from the same patient. TMA were stained with the FDA-approved MRQ-48 antibody against TfR and graded by staining intensity (H-score). Patient-derived xenografts (PDX) and isogenic metastatic mouse models were used to study in vivo TfR expression and uptake of transferrin. RESULTS: TFRC gene and protein expression were high in breast cancer of all subtypes and stages, and in 60-85% of bone metastases. TfR was detectable after neoadjuvant chemotherapy, albeit with some variability. Fluorophore-conjugated transferrin iron chelator deferoxamine (DFO) enhanced TfR uptake in human breast cancer cells in vitro and proved transferrin localization at metastatic sites and correlation of tumor burden relative to untreated tumor mice. CONCLUSIONS: TfR is expressed in breast cancer, primary, metastatic, and after neoadjuvant chemotherapy. Variability in expression of TfR suggests that evaluation of the expression of TfR in individual patients could identify the best candidates for targeting. Further, systemic iron chelation with DFO may upregulate receptor expression and improve uptake of therapeutics or tracers that use transferrin as a homing ligand.

Frequency of Hepatic Metastatic Disease in Patients with Stage IV Breast Cancer Is Similar for Steatotic and Non-Steatotic Livers.

Background: Breast cancer is the most common non-cutaneous malignancy and the second leading cause of cancer mortality in the United States. Breast cancer is a heterogeneous disease; diagnosis at an early stage renders it potentially curable, whereas advanced metastatic disease carries a worse prognosis. Objectives: To investigate whether hepatic steatosis (HS) is associated with liver metastases in patients with newly diagnosed stage IV female breast cancer patients (either de novo metastatic breast cancer or recurrent metastatic breast cancer) using non-contrast computed tomography (CT) as a marker of HS. Design: Retrospective analysis. Methods: We retrospectively identified 168 patients with stage IV breast cancer with suitable imaging from a prospectively maintained oncologic database. Three radiologists manually defined hepatic regions of interest on non-contrast CT images, and attenuation data were extracted. HS was defined as a mean attenuation <48 Hounsfield units. The frequency of hepatic metastatic disease was calculated for patient with and without HS. Relationships between HS and various patient (age, body mass index, race) and tumor (hormone receptor status, HER2 status, tumor grade) characteristics were also analyzed. Results: There were 4 patients with liver metastasis in the HS group (41 patients) versus 20 patients with liver metastases in the non-HS group (127 patients). The difference in frequencies of liver metastases among patients with (9.8%) versus without (15.7%) hepatic steatosis (odds ratio = 1.72 [0.53-7.39]) was not statistically significant (P = .45). Body mass index was significantly higher (P = .01) among patients with hepatic steatosis (32.2 ± 7.3 vs 28.8 ± 7.1 kg/m2). Otherwise, there were no significant differences between patients with versus without HS with respect to regarding age, race, hormone receptor status, HER2 status, or tumor grade. Conclusion: The frequency of hepatic metastatic disease in patients with stage IV breast cancer is similar for steatotic and non-steatotic livers.

p38MAPKα Stromal Reprogramming Sensitizes Metastatic Breast Cancer to Immunotherapy.

Metastatic breast cancer is an intractable disease that responds poorly to immunotherapy. We show that p38MAPKα inhibition (p38i) limits tumor growth by reprogramming the metastatic tumor microenvironment in a CD4+ T cell-, IFNγ-, and macrophage-dependent manner. To identify targets that further increased p38i efficacy, we utilized a stromal labeling approach and single-cell RNA sequencing. Thus, we combined p38i and an OX40 agonist that synergistically reduced metastatic growth and increased overall survival. Intriguingly, patients with a p38i metastatic stromal signature had better overall survival that was further improved by the presence of an increased mutational load, leading us to ask if our approach would be effective in antigenic breast cancer. The combination of p38i, anti-OX40, and cytotoxic T-cell engagement cured mice of metastatic disease and produced long-term immunologic memory. Our findings demonstrate that a detailed understanding of the stromal compartment can be used to design effective antimetastatic therapies. SIGNIFICANCE: Immunotherapy is rarely effective in breast cancer. We dissected the metastatic tumor stroma, which revealed a novel therapeutic approach that targets the stromal p38MAPK pathway and creates an opportunity to unleash an immunologic response. Our work underscores the importance of understanding the tumor stromal compartment in therapeutic design. This article is highlighted in the In This Issue feature, p. 1275.

A phase II study of palbociclib plus letrozole plus trastuzumab as neoadjuvant treatment for clinical stages II and III ER+ HER2+ breast cancer (PALTAN).

Patients with ER+/HER2+ breast cancer (BC) are less likely to achieve pathological complete response (pCR) after chemotherapy with dual HER2 blockade than ER-/HER2+ BC. Endocrine therapy plus trastuzumab is effective in advanced ER+/HER2+ BC. Inhibition of CDK4/6 and HER2 results in synergistic cell proliferation reduction. We combined palbociclib, letrozole, and trastuzumab (PLT) as a chemotherapy-sparing regimen. We evaluated neoadjuvant PLT in early ER+/HER2+ BC. Primary endpoint was pCR after 16 weeks. Research biopsies were performed for whole exome and RNA sequencing, PAM50 subtyping, and Ki67 assessment for complete cell cycle arrest (CCCA: Ki67 ≤ 2.7%). After 26 patients, accrual stopped due to futility. pCR (residual cancer burden-RCB 0) was 7.7%, RCB 0/I was 38.5%. Grade (G) 3/4 treatment-emergent adverse events occurred in 19. Among these, G3/4 neutropenia was 50%, hypertension 26.9%, and leucopenia 7.7%. Analysis indicated CCCA in 85% at C1 day 15 (C1D15), compared to 27% at surgery after palbociclib was discontinued. Baseline PAM50 subtyping identified 31.2% HER2-E, 43.8% Luminal B, and 25% Luminal A. 161 genes were differentially expressed comparing C1D15 to baseline. MKI67, TK1, CCNB1, AURKB, and PLK1 were among the genes downregulated, consistent with CCCA at C1D15. Molecular Signatures Database gene-sets analyses demonstrated downregulated processes involved in proliferation, ER and mTORC1 signaling, and DNA damage repair at C1D15, consistent with the study drug's mechanisms of action. Neoadjuvant PLT showed a pCR of 7.7% and an RCB 0/I rate of 38.5%. RNA sequencing and Ki67 data indicated potent anti-proliferative effects of study treatments. ClinicalTrials.gov- NCT02907918.

Genomic Complexity Predicts Resistance to Endocrine Therapy and CDK4/6 Inhibition in Hormone Receptor-Positive (HR+)/HER2-Negative Metastatic Breast Cancer.

PURPOSE: Clinical biomarkers to identify patients unlikely to benefit from CDK4/6 inhibition (CDK4/6i) in combination with endocrine therapy (ET) are lacking. We implemented a comprehensive circulating tumor DNA (ctDNA) analysis to identify genomic features for predicting and monitoring treatment resistance. EXPERIMENTAL DESIGN: ctDNA was isolated from 216 plasma samples collected from 51 patients with hormone receptor-positive (HR+)/HER2-negative (HER2-) metastatic breast cancer (MBC) on a phase II trial of palbociclib combined with letrozole or fulvestrant (NCT03007979). Boosted whole-exome sequencing (WES) was performed at baseline and clinical progression to evaluate genomic alterations, mutational signatures, and blood tumor mutational burden (bTMB). Low-pass whole-genome sequencing was performed at baseline and serial timepoints to assess blood copy-number burden (bCNB). RESULTS: High bTMB and bCNB were associated with lack of clinical benefit and significantly shorter progression-free survival (PFS) compared with patients with low bTMB or low bCNB (all P < 0.05). Dominant APOBEC signatures were detected at baseline exclusively in cases with high bTMB (5/13, 38.5%) versus low bTMB (0/37, 0%; P = 0.0006). Alterations in ESR1 were enriched in samples with high bTMB (P = 0.0005). There was a high correlation between bTMB determined by WES and bTMB determined using a 600-gene panel (R = 0.98). During serial monitoring, an increase in bCNB score preceded radiographic progression in 12 of 18 (66.7%) patients. CONCLUSIONS: Genomic complexity detected by noninvasive profiling of bTMB and bCNB predicted poor outcomes in patients treated with ET and CDK4/6i and identified early disease progression before imaging. Novel treatment strategies including immunotherapy-based combinations should be investigated in this population.

Yoga Therapy During Chemotherapy for Early-Stage and Locally Advanced Breast Cancer.

BACKGROUND: Chemotherapy is associated with decreased quality of life (QOL), fatigue, depression, and weight gain in patients with breast cancer. Weight gain is associated with poorer prognosis. Yoga improves QOL, fatigue, and mood in women with breast cancer but its effect on treatment-related weight gain has not been studied. The aim of this trial was to determine the feasibility of personalized yoga therapy in women receiving treatment for early-stage or locally advanced breast cancer and assess its impact on weight gain. METHODS: Thirty women were randomized 1:1 to receive yoga therapy by a certified yoga therapist during treatment or a control group. Participants in the yoga arm were asked to complete three 30 minute yoga sessions weekly (which included movement, breath work, mindfulness, and relaxation) throughout adjuvant or neoadjuvant chemotherapy (N = 29) or endocrine (N = 1); the control arm received breast cancer treatment without yoga. For comparability between participants randomized to yoga therapy, the single patient treated with endocrine therapy was excluded from the analysis. Primary outcomes were feasibility and weight change. Additional outcomes were mood, fatigue, QOL, serum tumor necrosis factor-alpha (TNF-alpha), and C-reactive protein (CRP) as immune mediator biomarkers. RESULTS: Mean age was 51.6 years, 75.9% were white and 24.1% were people of color, reflecting the cancer center population. 80% had stage II-III disease. Enrollment was completed in 9 months. Compliance was lower than predicted; however, participants participated in on average 1.7 yoga sessions/week for a mean 15.6 weeks duration. There were no adverse events. Control arm participants gained on average 2.63% body weight during treatment while yoga participants lost 0.14% body weight (weight change = -0.36 in yoga arm vs. 2.89 in standard of care arm, Wilcoxon rank sum test P = .024). Control participants reported increased fatigue and decreased QOL, while yoga participants reported no change in QOL. No significant change in TNF-alpha or CRP was noted in either arm. CONCLUSION: This feasibility study suggests that personalized yoga therapy is beneficial for QOL and weight maintenance among women undergoing chemotherapy for early-stage or locally advanced breast cancer. Weight maintenance associated with yoga therapy may be of clinical significance in this population given the poorer prognosis associated with weight gain in breast cancer survivors. TRIAL REGISTRATION: NIH Clinicaltrials.gov #NCT03262831; August 25, 2017. https://clinicaltrials.gov/ct2/show/NCT03262831.

A randomized feasibility study evaluating temozolomide circadian medicine in patients with glioma.

Background: Gliomas are the most common primary brain tumor in adults. Current treatments involve surgery, radiation, and temozolomide (TMZ) chemotherapy; however, prognosis remains poor and new approaches are required. Circadian medicine aims to maximize treatment efficacy and/or minimize toxicity by timed delivery of medications in accordance with the daily rhythms of the patient. We published a retrospective study showing greater anti-tumor efficacy for the morning, relative to the evening, administration of TMZ in patients with glioblastoma. We conducted this prospective randomized trial to determine the feasibility, and potential clinical impact, of TMZ chronotherapy in patients with gliomas (NCT02781792). Methods: Adult patients with gliomas (WHO grade II-IV) were enrolled prior to initiation of monthly TMZ therapy and were randomized to receive TMZ either in the morning (AM) before 10 am or in the evening (PM) after 8 pm. Pill diaries were recorded to measure compliance and FACT-Br quality of life (QoL) surveys were completed throughout treatment. Study compliance, adverse events (AE), and overall survival were compared between the two arms. Results: A total of 35 evaluable patients, including 21 with GBM, were analyzed (18 AM patients and 17 PM patients). Compliance data demonstrated the feasibility of timed TMZ dosing. There were no significant differences in AEs, QoL, or survival between the arms. Conclusions: Chronotherapy with TMZ is feasible. A larger study is needed to validate the effect of chronotherapy on clinical efficacy.

A phase II trial of an alternative schedule of palbociclib and embedded serum TK1 analysis.

Palbociclib 3-weeks-on/1-week-off, combined with hormonal therapy, is approved for hormone receptor positive (HR+)/HER2-negative (HER2-) advanced/metastatic breast cancer (MBC). Neutropenia is the most frequent adverse event (AE). We aim to determine whether an alternative 5-days-on/2-days-off weekly schedule reduces grade 3 and above neutropenia (G3 + ANC) incidence. In this single-arm phase II trial, patients with HR+/HER2- MBC received palbociclib 125 mg, 5-days-on/2-days-off, plus letrozole or fulvestrant per physician, on a 28-day cycle (C), as their first- or second-line treatment. The primary endpoint was G3 + ANC in the first 29 days (C1). Secondary endpoints included AEs, efficacy, and serum thymidine kinase 1 (sTK1) activity. At data-cutoff, fifty-four patients received a median of 13 cycles (range 2.6-43.5). The rate of G3 + ANC was 21.3% (95% CI: 11.2-36.1%) without G4 in C1, and 40.7% (95% CI: 27.9-54.9%), including 38.9% G3 and 1.8% G4, in all cycles. The clinical benefit rate was 80.4% (95% CI: 66.5-89.7%). The median progression-free survival (mPFS) (95% CI) was 19.75 (12.11-34.89), 33.5 (17.25-not reached [NR]), and 11.96 (10.43-NR) months, in the overall, endocrine sensitive or resistant population, respectively. High sTK1 at baseline, C1 day 15 (C1D15), and C2D1 were independently prognostic for shorter PFS (p = 9.91 × 10-4, 0.001, 0.007, respectively). sTK1 decreased on C1D15 (p = 4.03 × 10-7), indicating target inhibition. Rise in sTK1 predicted progression, with the median lead time of 59.5 (inter-quartile range: -206.25-0) days. Palbociclib, 5-days-on/2-days-off weekly, met its primary endpoint with reduced G3 + ANC, without compromising efficacy. sTK1 is prognostic and shows promise in monitoring the palbociclib response. ClinicalTrials.gov#: NCT3007979.

Heparanase Blockade as a Novel Dual-Targeting Therapy for COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused over 5 million deaths worldwide. Pneumonia and systemic inflammation contribute to its high mortality. Many viruses use heparan sulfate proteoglycans as coreceptors for viral entry, and heparanase (HPSE) is a known regulator of both viral entry and inflammatory cytokines. We evaluated the heparanase inhibitor Roneparstat, a modified heparin with minimum anticoagulant activity, in pathophysiology and therapy for COVID-19. We found that Roneparstat significantly decreased the infectivity of SARS-CoV-2, SARS-CoV-1, and retroviruses (human T-lymphotropic virus 1 [HTLV-1] and HIV-1) in vitro. Single-cell RNA sequencing (scRNA-seq) analysis of cells from the bronchoalveolar lavage fluid of COVID-19 patients revealed a marked increase in HPSE gene expression in CD68+ macrophages compared to healthy controls. Elevated levels of HPSE expression in macrophages correlated with the severity of COVID-19 and the expression of inflammatory cytokine genes, including IL6, TNF, IL1B, and CCL2. In line with this finding, we found a marked induction of HPSE and numerous inflammatory cytokines in human macrophages challenged with SARS-CoV-2 S1 protein. Treatment with Roneparstat significantly attenuated SARS-CoV-2 S1 protein-mediated inflammatory cytokine release from human macrophages, through disruption of NF-κB signaling. HPSE knockdown in a macrophage cell line also showed diminished inflammatory cytokine production during S1 protein challenge. Taken together, this study provides a proof of concept that heparanase is a target for SARS-CoV-2-mediated pathogenesis and that Roneparstat may serve as a dual-targeted therapy to reduce viral infection and inflammation in COVID-19. IMPORTANCE The complex pathogenesis of COVID-19 consists of two major pathological phases: an initial infection phase elicited by SARS-CoV-2 entry and replication and an inflammation phase that could lead to tissue damage, which can evolve into acute respiratory failure or even death. While the development and deployment of vaccines are ongoing, effective therapy for COVID-19 is still urgently needed. In this study, we explored HPSE blockade with Roneparstat, a phase I clinically tested HPSE inhibitor, in the context of COVID-19 pathogenesis. Treatment with Roneparstat showed wide-spectrum anti-infection activities against SARS-CoV-2, HTLV-1, and HIV-1 in vitro. In addition, HPSE blockade with Roneparstat significantly attenuated SARS-CoV-2 S1 protein-induced inflammatory cytokine release from human macrophages through disruption of NF-κB signaling. Together, this study provides a proof of principle for the use of Roneparstat as a dual-targeting therapy for COVID-19 to decrease viral infection and dampen the proinflammatory immune response mediated by macrophages.

Machine learning-based scoring models to predict hematopoietic stem cell mobilization in allogeneic donors.

Mobilized peripheral blood has become the primary source of hematopoietic stem cells for both autologous and allogeneic stem cell transplantation. Granulocyte colony-stimulating factor (G-CSF) is currently the standard agent used in the allogeneic setting. Despite the high mobilization efficacy in most donors, G-CSF requires 4-5 days of daily administration, and a small percentage of the donors fail to mobilize an optimal number of stem cells necessary for a safe allogeneic stem cell transplant. In this study, we retrospectively reviewed 1361 related allogeneic donors who underwent stem cell mobilization at Washington University. We compared the standard mobilization agent G-CSF with five alternative mobilization regimens, including GM-CSF, G-CSF+GM-CSF, GM-CSF + Plerixafor, Plerixafor and BL-8040. Cytokine-based mobilization strategies (G-CSF or in combination with GM-CSF) induce higher CD34 cell yield after 4-5 consecutive days of treatment, while CXCR4 antagonists (plerixafor and BL-8040) induce significantly less but rapid mobilization on the same day. Next, using a large dataset containing the demographic and baseline laboratory data from G-CSF-mobilized donors, we established machine learning (ML)-based scoring models that can be used to predict patients who may have less than optimal stem cell yields after a single leukapheresis session. To our knowledge, this is the first prediction model at the early donor screening stage, which may help identify allogeneic stem cell donors who may benefit from alternative approaches to enhance stem cell yields, thus ensuring safe and effective stem cell transplantation.

Organ-on-a-chip model of vascularized human bone marrow niches.

Bone marrow niches (endosteal and perivascular) play important roles in both normal bone marrow function and pathological processes such as cancer cell dormancy. Unraveling the mechanisms underlying these events in humans has been severely limited by models that cannot dissect dynamic events at the niche level. Utilizing microfluidic and stem cell technologies, we present a 3D in vitro model of human bone marrow that contains both the perivascular and endosteal niches, complete with dynamic, perfusable vascular networks. We demonstrate that our model can replicate in vivo bone marrow function, including maintenance and differentiation of CD34+ hematopoietic stem/progenitor cells, egress of neutrophils (CD66b+), and niche-specific responses to doxorubicin and granulocyte-colony stimulating factor. Our platform provides opportunities to accelerate current understanding of human bone marrow function and drug response with high spatial and temporal resolution.

Genetic Counseling and Testing in African American Patients With Breast Cancer: A Nationwide Survey of US Breast Oncologists.

PURPOSE: To determine if physicians' self-reported knowledge, attitudes, and practices regarding genetic counseling and testing (GCT) vary by patients' race. METHODS: We conducted a nationwide 49-item survey among breast oncology physicians in the United States. We queried respondents about their own demographics, clinical characteristics, knowledge, attitudes, practices, and perceived barriers in providing GCT to patients with breast cancer. RESULTS: Our survey included responses from 277 physicians (females, 58.8%; medical oncologists, 75.1%; academic physicians, 61.7%; and Whites, 67.1%). Only 1.8% indicated that they were more likely to refer a White patient than refer an African American patient for GCT, and 66.9% believed that African American women with breast cancer have lower rates of GCT than White women. Regarding perceived barriers to GCT, 63.4% of respondents indicated that African American women face more barriers than White women do and 21% felt that African American women require more information and guidance during the GCT decision-making process than White women. Although 32% of respondents indicated that lack of trust was a barrier to GCT in all patients, 58.1% felt that this was a greater barrier for African American women (P < .0001). Only 13.9% believed that noncompliance with GCT is a barrier for all patients, whereas 30.6% believed that African American women are more likely than White women to be noncompliant (P < .0001). CONCLUSION: We demonstrated that racial differences exist in oncology physicians' perceived barriers to GCT for patients with breast cancer. This nationwide survey will serve as a basis for understanding physicians' determinants of GCT for African American women and highlights the necessity of education and interventions to address bias among physicians. Awareness of such physician biases can enable further work to address inequities, ultimately leading to improved GCT equity for African American women with breast cancer.

Breast cancer-derived GM-CSF regulates arginase 1 in myeloid cells to promote an immunosuppressive microenvironment.

Tumor-infiltrating myeloid cells contribute to the development of the immunosuppressive tumor microenvironment. Myeloid cell expression of arginase 1 (ARG1) promotes a protumor phenotype by inhibiting T cell function and depleting extracellular l-arginine, but the mechanism underlying this expression, especially in breast cancer, is poorly understood. In breast cancer clinical samples and in our mouse models, we identified tumor-derived GM-CSF as the primary regulator of myeloid cell ARG1 expression and local immune suppression through a gene-KO screen of breast tumor cell-produced factors. The induction of myeloid cell ARG1 required GM-CSF and a low pH environment. GM-CSF signaling through STAT3 and p38 MAPK and acid signaling through cAMP were required to activate myeloid cell ARG1 expression in a STAT6-independent manner. Importantly, breast tumor cell-derived GM-CSF promoted tumor progression by inhibiting host antitumor immunity, driving a significant accumulation of ARG1-expressing myeloid cells compared with lung and melanoma tumors with minimal GM-CSF expression. Blockade of tumoral GM-CSF enhanced the efficacy of tumor-specific adoptive T cell therapy and immune checkpoint blockade. Taken together, we show that breast tumor cell-derived GM-CSF contributes to the development of the immunosuppressive breast cancer microenvironment by regulating myeloid cell ARG1 expression and can be targeted to enhance breast cancer immunotherapy.

Antibiotic-induced disturbances of the gut microbiota result in accelerated breast tumor growth.

The gut microbiota's function in regulating health has seen it linked to disease progression in several cancers. However, there is limited research detailing its influence in breast cancer (BrCa). This study found that antibiotic-induced perturbation of the gut microbiota significantly increases tumor progression in multiple BrCa mouse models. Metagenomics highlights the common loss of several bacterial species following antibiotic administration. One such bacteria, Faecalibaculum rodentium, rescued this increased tumor growth. Single-cell transcriptomics identified an increased number of cells with a stromal signature in tumors, and subsequent histology revealed an increased abundance of mast cells in the tumor stromal regions. We show that administration of a mast cell stabilizer, cromolyn, rescues increased tumor growth in antibiotic treated animals but has no influence on tumors from control cohorts. These findings highlight that BrCa-microbiota interactions are different from other cancers studied to date and suggest new research avenues for therapy development.

Disseminated tumour cells from the bone marrow of early breast cancer patients: Results from an international pooled analysis.

PURPOSE: Presence of disseminated tumour cells (DTCs) in the bone marrow (BM) has been described as a surrogate of residual disease in patients with early breast cancer (EBC). PADDY (Pooled Analysis of DTC Detection in Early Breast Cancer) is a large international analysis of pooled data that aimed to assess the prognostic impact of DTCs in patients with EBC. EXPERIMENTAL DESIGN: Individual patient data were collected from 11 centres. Patients with EBC and available follow-up data in whom BM sampling was performed at the time of primary diagnosis before receiving any anticancer treatment were eligible. DTCs were identified by antibody staining against epithelial cytokeratins. Multivariate Cox regression was used to compare the survival of DTC-positive versus DTC-negative patients. RESULTS: In total, 10,307 patients were included. Of these, 2814 (27.3%) were DTC-positive. DTC detection was associated with higher tumour grade, larger tumour size, nodal positivity, oestrogen receptor and progesterone receptor negativity, and HER2 positivity (all p < 0.001). Multivariate analyses showed that DTC detection was an independent prognostic marker for overall survival, disease-free survival and distant disease-free survival with hazard ratios (HR) and 95% confidence intervals (CI) of 1.23 (95% CI: 1.06-1.43, p = 0.006), 1.30 (95% CI: 1.12-1.52, p < 0.001) and 1.30 (95% CI: 1.08-1.56, p = 0.006), respectively. There was no association between locoregional relapse-free survival and DTC detection (HR 1.21; 95% CI 0.68-2.16; p = 0.512). CONCLUSIONS: DTCs in the BM represent an independent prognostic marker in patients with EBC. The heterogeneous metastasis-initiating potential of DTCs is consistent with the concept of cancer dormancy.