Safety and efficacy of C. muridarum vaccines adjuvanted with CpG-1826 and four concentrations of Montanide-ISA-720-VG.

It is recommended that the adjuvant Montanide ISA 720 VG be used at a concentration of 70% v/v. At this concentration, Montanide causes at the site of immunization a local granuloma that can last for several weeks. To determine the safety and protective efficacy of a Chlamydia muridarum MOMP vaccine, formulated with CpG-1826 and four different concentrations of Montanide (70%, 50%, 30% and 10%), BALB/c (H-2d) female mice were immunized twice intramuscularly. Local reactogenicity was significant for vaccines formulated with 70% or 50% Montanide but not for those inoculated with 30% or 10% Montanide. Robust humoral and cell mediated memory immune responses were elicited by the 70%, 50% and 30% Montanide formulations. Mice were challenged intranasally with 104 C. muridarum inclusion forming units (IFU). Based on changes in body weight, lungs's weight and number of IFU recovered, mice vaccinated with the 70%, 50% and 30% Montanide formulations were significantly protected, but not mice receiving 10% Montanide. To conclude, we recommend the 30% Montanide concentration to be tested in humans and animal models to determine its safety and efficacy, in comparison to the 70% Montanide concentration currently used. The 30% Montanide formulation could significantly facilitate licensing of this adjuvant for human use.

Loss of Cadherin-11 in pancreatic ductal adenocarcinoma alters tumor-immune microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is one of the top five deadliest forms of cancer with very few treatment options. The 5-year survival rate for PDAC is 10% following diagnosis. Cadherin 11 (Cdh11), a cell-to-cell adhesion molecule, has been suggested to promote tumor growth and immunosuppression in PDAC, and Cdh11 inhibition significantly extended survival in mice with PDAC. However, the mechanisms by which Cdh11 deficiency influences PDAC progression and anti-tumor immune responses have yet to be fully elucidated. To investigate Cdh11-deficiency induced changes in PDAC tumor microenvironment (TME), we crossed p48-Cre; LSL-KrasG12D/+; LSL-Trp53R172H/+ (KPC) mice with Cdh11+/- mice and performed single-cell RNA sequencing (scRNA-seq) of the non-immune (CD45-) and immune (CD45+) compartment of KPC tumor-bearing Cdh11 proficient (KPC-Cdh11+/+) and Cdh11 deficient (KPC-Cdh11+/-) mice. Our analysis showed that Cdh11 is expressed primarily in cancer-associated fibroblasts (CAFs) and at low levels in epithelial cells undergoing epithelial-to-mesenchymal transition (EMT). Cdh11 deficiency altered the molecular profile of CAFs, leading to a decrease in the expression of myofibroblast markers such as Acta2 and Tagln and cytokines such as Il6, Il33 and Midkine (Mdk). We also observed a significant decrease in the presence of monocytes/macrophages and neutrophils in KPC-Cdh11+/- tumors while the proportion of T cells was increased. Additionally, myeloid lineage cells from Cdh11-deficient tumors had reduced expression of immunosuppressive cytokines that have previously been shown to play a role in immune suppression. In summary, our data suggests that Cdh11 deficiency significantly alters the fibroblast and immune microenvironments and contributes to the reduction of immunosuppressive cytokines, leading to an increase in anti-tumor immunity and enhanced survival.

A perfused multi-well bioreactor platform to assess tumor organoid response to a chemotherapeutic gradient.

There is an urgent need to develop new therapies for colorectal cancer that has metastasized to the liver and, more fundamentally, to develop improved preclinical platforms of colorectal cancer liver metastases (CRCLM) to screen therapies for efficacy. To this end, we developed a multi-well perfusable bioreactor capable of monitoring CRCLM patient-derived organoid response to a chemotherapeutic gradient. CRCLM patient-derived organoids were cultured in the multi-well bioreactor for 7 days and the subsequently established gradient in 5-fluorouracil (5-FU) concentration resulted in a lower IC50 in the region near the perfusion channel versus the region far from the channel. We compared behaviour of organoids in this platform to two commonly used PDO culture models: organoids in media and organoids in a static (no perfusion) hydrogel. The bioreactor IC50 values were significantly higher than IC50 values for organoids cultured in media whereas only the IC50 for organoids far from the channel were significantly different than organoids cultured in the static hydrogel condition. Using finite element simulations, we showed that the total dose delivered, calculated using area under the curve (AUC) was similar between platforms, however normalized viability was lower for the organoid in media condition than in the static gel and bioreactor. Our results highlight the utility of our multi-well bioreactor for studying organoid response to chemical gradients and demonstrate that comparing drug response across these different platforms is nontrivial.

Modulation of Radiation Biomarkers in a Randomized Phase II Study of 131I-MIBG With or Without Radiation Sensitizers for Relapsed or Refractory Neuroblastoma.

PURPOSE: 131I-metaiodobenzylguanidine (131I-MIBG) has demonstrated efficacy as a single agent in neuroblastoma. Recent trials have focused on 131I-MIBG combination strategies, though little is known about the effect of putative radiosensitizers on biological markers of radiation exposure. METHODS AND MATERIALS: NANT2011-01 evaluated 131I-MIBG therapy alone (arm A) or in combination with vincristine/irinotecan (arm B) or vorinostat (arm C) for patients with relapsed or refractory neuroblastoma. Blood samples were collected before and after 131I-MIBG infusion to determine levels of radiation-associated biomarkers (transcript and protein). The association of biomarker with treatment arm, clinical response, and treatment toxicity was analyzed. RESULTS: The cohort included 99 patients who had at least 1 biomarker available for analysis. Significant modulation in most biomarkers between baseline, 72, and 96 hours following 131I-MIBG was observed. Patients in arm C had the lowest degree of modulation in FLT3 ligand protein. Lower baseline BCL2 transcript levels were associated with higher overall response. Patients with greater increases in FLT3 ligand at 96 hours after 131I-MIBG therapy were significantly more likely to have grade 4 thrombocytopenia. Peripheral blood gene expression of the BCL2 family of apoptotic markers (BCL2L1 and BAX transcripts) was significantly associated with grade 4 hematologic toxicity. RNA sequencing demonstrated little overlap in the top modulated peripheral blood transcripts between randomized arms. CONCLUSIONS: Peripheral blood biomarkers relevant to radiation exposure demonstrate significant modulation after 131I-MIBG and concomitant radiation sensitizers affect extent of modulation. Biomarkers related to hematopoietic damage and apoptosis were associated with hematologic toxicity.

Immediate effects of acute Mars mission equivalent doses of SEP and GCR radiation on the murine gastrointestinal system-protective effects of curcumin-loaded nanolipoprotein particles (cNLPs).

Introduction: Missions beyond low Earth orbit (LEO) will expose astronauts to ionizing radiation (IR) in the form of solar energetic particles (SEP) and galactic cosmic rays (GCR) including high atomic number and energy (HZE) nuclei. The gastrointestinal (GI) system is documented to be highly radiosensitive with even relatively low dose IR exposures capable of inducing mucosal lesions and disrupting epithelial barrier function. IR is also an established risk factor for colorectal cancer (CRC) with several studies examining long-term GI effects of SEP/GCR exposure using tumor-prone APC mouse models. Studies of acute short-term effects of modeled space radiation exposures in wildtype mouse models are more limited and necessary to better define charged particle-induced GI pathologies and test novel medical countermeasures (MCMs) to promote astronaut safety. Methods: In this study, we performed ground-based studies where male and female C57BL/6J mice were exposed to γ-rays, 50 MeV protons, or 1 GeV/n Fe-56 ions at the NASA Space Radiation Laboratory (NSRL) with histology and immunohistochemistry endpoints measured in the first 24 h post-irradiation to define immediate SEP/GCR-induced GI alterations. Results: Our data show that unlike matched γ-ray controls, acute exposures to protons and iron ions disrupts intestinal function and induces mucosal lesions, vascular congestion, epithelial barrier breakdown, and marked enlargement of mucosa-associated lymphoid tissue. We also measured kinetics of DNA double-strand break (DSB) repair using gamma-H2AX- specific antibodies and apoptosis via TUNEL labeling, noting the induction and disappearance of extranuclear cytoplasmic DNA marked by gamma-H2AX only in the charged particle-irradiated samples. We show that 18 h pre-treatment with curcumin-loaded nanolipoprotein particles (cNLPs) delivered via IV injection reduces DSB-associated foci levels and apoptosis and restore crypt villi lengths. Discussion: These data improve our understanding of physiological alterations in the GI tract immediately following exposures to modeled space radiations and demonstrates effectiveness of a promising space radiation MCM.

PERIOD 2 regulates low-dose radioprotection via PER2/pGSK3ß/ß-catenin/Per2 loop.

During evolution, humans are acclimatized to the stresses of natural radiation and circadian rhythmicity. Radiosensitivity of mammalian cells varies in the circadian period and adaptive radioprotection can be induced by pre-exposure to low-level radiation (LDR). It is unclear, however, if clock proteins participate in signaling LDR radioprotection. Herein, we demonstrate that radiosensitivity is increased in mice with the deficient Period 2 gene (Per2def) due to impaired DNA repair and mitochondrial function in progenitor bone marrow hematopoietic stem cells and monocytes. Per2 induction and radioprotection are also identified in LDR-treated Per2wt mouse cells and in human skin (HK18) and breast (MCF-10A) epithelial cells. LDR-boosted PER2 interacts with pGSK3ß(S9) which activates ß-catenin and the LEF/TCF mediated gene transcription including Per2 and genes involved in DNA repair and mitochondrial functions. This study demonstrates that PER2 plays an active role in LDR adaptive radioprotection via PER2/pGSK3ß/ß-catenin/Per2 loop, a potential target for protecting normal cells from radiation injury.

Curcumin Nanodiscs Improve Solubility and Serve as Radiological Protectants against Ionizing Radiation Exposures in a Cell-Cycle Dependent Manner.

Curcumin, a natural polyphenol derived from the spice turmeric (Curcuma longa), contains antioxidant, anti-inflammatory, and anti-cancer properties. However, curcumin bioavailability is inherently low due to poor water solubility and rapid metabolism. Here, we further refined for use curcumin incorporated into "biomimetic" nanolipoprotein particles (cNLPs) consisting of a phospholipid bilayer surrounded by apolipoprotein A1 and amphipathic polymer scaffolding moieties. Our cNLP formulation improves the water solubility of curcumin over 30-fold and produces nanoparticles with ~350 µg/mL total loading capacity for downstream in vitro and in vivo applications. We found that cNLPs were well tolerated in AG05965/MRC-5 human primary lung fibroblasts compared to cultures treated with curcumin solubilized in DMSO (curDMSO). Pre-treatment with cNLPs of quiescent G0/G1-phase MRC-5 cultures improved cell survival following 137Cs gamma ray irradiations, although this finding was reversed in asynchronously cycling log-phase cell cultures. These findings may be useful for establishing cNLPs as a method to improve curcumin bioavailability for administration as a radioprotective and/or radiomitigative agent against ionizing radiation (IR) exposures in non-cycling cells or as a radiosensitizing agent for actively dividing cell populations, such as tumors.

IL-17A Increases Doxorubicin Efficacy in Triple Negative Breast Cancer.

Due to lack of targetable receptors and intertumoral heterogeneity, triple negative breast cancer (TNBC) remains particularly difficult to treat. Doxorubicin (DOX) is typically used as nonselective neoadjuvant chemotherapy, but the diversity of treatment efficacy remains unclear. Comparable to variability in clinical response, an experimental model of TNBC using a 4T1 syngeneic mouse model was found to elicit a differential response to a seven-day treatment regimen of DOX. Single-cell RNA sequencing identified an increase in T cells in tumors that responded to DOX treatment compared to tumors that continued to grow uninhibited. Additionally, compared to resistant tumors, DOX sensitive tumors contained significantly more CD4 T helper cells (339%), γδ T cells (727%), Naïve T cells (278%), and activated CD8 T cells (130%). Furthermore, transcriptional profiles of tumor infiltrated T cells in DOX responsive tumors revealed decreased exhaustion, increased chemokine/cytokine expression, and increased activation and cytotoxic activity. γδ T cell derived IL-17A was identified to be highly abundant in the sensitive tumor microenvironment. IL-17A was also found to directly increase sensitivity of TNBC cells in combination with DOX treatment. In TNBC tumors sensitive to DOX, increased IL-17A levels lead to a direct effect on cancer cell responsiveness and chronic stimulation of tumor infiltrated T cells leading to improved chemotherapeutic efficacy. IL-17A's role as a chemosensitive cytokine in TNBC may offer new opportunities for treating chemoresistant breast tumors and other cancer types.

Spaceflight-Associated Changes of snoRNAs in Peripheral Blood Mononuclear Cells and Plasma Exosomes-A Pilot Study.

During spaceflight, astronauts are exposed to various physiological and psychological stressors that have been associated with adverse health effects. Therefore, there is an unmet need to develop novel diagnostic tools to predict early alterations in astronauts' health. Small nucleolar RNA (snoRNA) is a type of short non-coding RNA (60-300 nucleotides) known to guide 2'-O-methylation (Nm) or pseudouridine (ψ) of ribosomal RNA (rRNA), small nuclear RNA (snRNA), or messenger RNA (mRNA). Emerging evidence suggests that dysregulated snoRNAs may be key players in regulating fundamental cellular mechanisms and in the pathogenesis of cancer, heart, and neurological disease. Therefore, we sought to determine whether the spaceflight-induced snoRNA changes in astronaut's peripheral blood (PB) plasma extracellular vesicles (PB-EV) and peripheral blood mononuclear cells (PBMCs). Using unbiased small RNA sequencing (sRNAseq), we evaluated changes in PB-EV snoRNA content isolated from astronauts (n = 5/group) who underwent median 12-day long Shuttle missions between 1998 and 2001. Using stringent cutoff (fold change > 2 or log2-fold change >1, FDR < 0.05), we detected 21 down-and 9-up-regulated snoRNAs in PB-EVs 3 days after return (R + 3) compared to 10 days before launch (L-10). qPCR validation revealed that SNORA74A was significantly down-regulated at R + 3 compared to L-10. We next determined snoRNA expression levels in astronauts' PBMCs at R + 3 and L-10 (n = 6/group). qPCR analysis further confirmed a significant increase in SNORA19 and SNORA47 in astronauts' PBMCs at R + 3 compared to L-10. Notably, many downregulated snoRNA-guided rRNA modifications, including four Nms and five ψs. Our findings revealed that spaceflight induced changes in PB-EV and PBMCs snoRNA expression, thus suggesting snoRNAs may serve as potential novel biomarkers for monitoring astronauts' health.

Ligand-induced transmembrane conformational coupling in monomeric EGFR.

Single pass cell surface receptors regulate cellular processes by transmitting ligand-encoded signals across the plasma membrane via changes to their extracellular and intracellular conformations. This transmembrane signaling is generally initiated by ligand binding to the receptors in their monomeric form. While subsequent receptor-receptor interactions are established as key aspects of transmembrane signaling, the contribution of monomeric receptors has been challenging to isolate due to the complexity and ligand-dependence of these interactions. By combining membrane nanodiscs produced with cell-free expression, single-molecule Förster Resonance Energy Transfer measurements, and molecular dynamics simulations, we report that ligand binding induces intracellular conformational changes within monomeric, full-length epidermal growth factor receptor (EGFR). Our observations establish the existence of extracellular/intracellular conformational coupling within a single receptor molecule. We implicate a series of electrostatic interactions in the conformational coupling and find the coupling is inhibited by targeted therapeutics and mutations that also inhibit phosphorylation in cells. Collectively, these results introduce a facile mechanism to link the extracellular and intracellular regions through the single transmembrane helix of monomeric EGFR, and raise the possibility that intramolecular transmembrane conformational changes upon ligand binding are common to single-pass membrane proteins.

Fatty acid oxidation fuels glioblastoma radioresistance with CD47-mediated immune evasion.

Glioblastoma multiforme (GBM) remains the top challenge to radiotherapy with only 25% one-year survival after diagnosis. Here, we reveal that co-enhancement of mitochondrial fatty acid oxidation (FAO) enzymes (CPT1A, CPT2 and ACAD9) and immune checkpoint CD47 is dominant in recurrent GBM patients with poor prognosis. A glycolysis-to-FAO metabolic rewiring is associated with CD47 anti-phagocytosis in radioresistant GBM cells and regrown GBM after radiation in syngeneic mice. Inhibition of FAO by CPT1 inhibitor etomoxir or CRISPR-generated CPT1A-/-, CPT2-/-, ACAD9-/- cells demonstrate that FAO-derived acetyl-CoA upregulates CD47 transcription via NF-κB/RelA acetylation. Blocking FAO impairs tumor growth and reduces CD47 anti-phagocytosis. Etomoxir combined with anti-CD47 antibody synergizes radiation control of regrown tumors with boosted macrophage phagocytosis. These results demonstrate that enhanced fat acid metabolism promotes aggressive growth of GBM with CD47-mediated immune evasion. The FAO-CD47 axis may be targeted to improve GBM control by eliminating the radioresistant phagocytosis-proofing tumor cells in GBM radioimmunotherapy.

Extracellular matrix modulates T cell clearance of malignant cells in vitro.

Despite the success of T cell checkpoint therapies, breast cancers rarely express these immunotherapy markers and are believed to be largely "immune cold" with limited inflammation and immune activation. The reason for this limited immune activation remains poorly understood. We sought to determine whether extracellular matrix substrate could contribute to this limited immune activation. Specifically, we asked whether extracellular matrix could alter T cell cytotoxicity against malignant mammary gland carcinoma cells (MCC) in a setup designed to promote maximal T cell efficacy (i.e., rich media with abundant IL2, high ratio of T cells to MCC). We observed that T cell clearance of MCC varied from 0% in collagen 4 or 6 conditions to almost 100% in fibronectin or vitronectin. Transcriptomics revealed that T cell function was defective in MCC/T cell cocultures on collagen 4 (Col4), potentially corresponding to greater expression of cytokines MCC cultured in this environment. In contrast, transcriptomics revealed an effective, exhausted phenotype on vitronectin. The observation that Col4 induces T cell suppression suggests that targeting tumor-ECM interactions may permit new approaches for utilizing immunotherapy in tumors which do not provoke a strong immune response.

Predictive Radiation Oncology - A New NCI-DOE Scientific Space and Community.

With a widely attended virtual kickoff event on January 29, 2021, the National Cancer Institute (NCI) and the Department of Energy (DOE) launched a series of 4 interactive, interdisciplinary workshops-and a final concluding "World Café" on March 29, 2021-focused on advancing computational approaches for predictive oncology in the clinical and research domains of radiation oncology. These events reflect 3,870 human hours of virtual engagement with representation from 8 DOE national laboratories and the Frederick National Laboratory for Cancer Research (FNL), 4 research institutes, 5 cancer centers, 17 medical schools and teaching hospitals, 5 companies, 5 federal agencies, 3 research centers, and 27 universities. Here we summarize the workshops by first describing the background for the workshops. Participants identified twelve key questions-and collaborative parallel ideas-as the focus of work going forward to advance the field. These were then used to define short-term and longer-term "Blue Sky" goals. In addition, the group determined key success factors for predictive oncology in the context of radiation oncology, if not the future of all of medicine. These are: cross-discipline collaboration, targeted talent development, development of mechanistic mathematical and computational models and tools, and access to high-quality multiscale data that bridges mechanisms to phenotype. The workshop participants reported feeling energized and highly motivated to pursue next steps together to address the unmet needs in radiation oncology specifically and in cancer research generally and that NCI and DOE project goals align at the convergence of radiation therapy and advanced computing.

Peripheral Blood Transcript Signatures after Internal 131I-mIBG Therapy in Relapsed and Refractory Neuroblastoma Patients Identifies Early and Late Biomarkers of Internal 131I Exposures.

131I-metaiodobenzylguanidine (131I-mIBG) is a targeted radiation therapy developed for the treatment of advanced neuroblastoma. We have previously shown that this patient cohort can be used to predict absorbed dose associated with early 131I exposure, 72 h after treatment. We now expand these studies to identify gene expression differences associated with 131I-mIBG exposure 15 days after treatment. Total RNA from peripheral blood lymphocytes was isolated from 288 whole blood samples representing 59 relapsed or refractory neuroblastoma patients before and after 131I-mIBG treatment. We found that several transcripts predictive of early exposure returned to baseline levels by day 15, however, selected transcripts did not return to baseline. At 72 h, all 17 selected pathway-specific transcripts were differentially expressed. Transcripts CDKN1A (P < 0.000001), FDXR (P < 0.000001), DDB2 (P < 0.000001), and BBC3 (P < 0.000001) showed the highest up-regulation at 72 h after 131I-mIBG exposure, with mean log2 fold changes of 2.55, 2.93, 1.86 and 1.85, respectively. At day 15 after 131I-mIBG, 11 of the 17 selected transcripts were differentially expressed, with XPC, STAT5B, PRKDC, MDM2, POLH, IGF1R, and SGK1 displaying significant up-regulation at 72 h and significant down-regulation at day 15. Interestingly, transcripts FDXR (P = 0.01), DDB2 (P = 0.03), BCL2 (P = 0.003), and SESN1 (P < 0.0003) maintained differential expression 15 days after 131I-mIBG treatment. These results suggest that transcript levels for DNA repair, apoptosis, and ionizing radiation-induced cellular stress are still changing by 15 days after 131I-mIBG treatment. Our studies showcase the use of biodosimetry gene expression panels as predictive biomarkers following early (72 h) and late (15 days) internal 131I exposure. Our findings also demonstrate the utility of our transcript panel to differentiate exposed from non-exposed individuals up to 15 days after exposure from internal 131I.

Nanolipoprotein-Mediated Her2 Protein Transfection Induces Malignant Transformation in Human Breast Acinar Cultures.

Her2 overexpression is associated with an aggressive form of breast cancer and malignant transformation. We demonstrate in this work that nanolipoprotein particles (NLPs) synthesized in a cell-free manner can be used to transfer Her2 protein into the membrane of nonmalignant cells in 3D culture in a nontoxic and facile manner. With NLP-mediated Her2 protein delivery, we observed an increased probability of nonmalignant cells forming apolar nongrowth-arrested tumor-like structures. The NLP delivery system alone or Her2-NLPs plus the Her2 inhibitor trastuzumab showed no effect on the acinar organization rate, indicating that Her2 signaling is key to this process. Transcriptomics revealed essentially no effect of empty NLPs compared to untreated cells, whereas Her2-NLPs versus either untreated or empty-NLP-treated cells revealed upregulation of several factors associated with breast cancer. Pathway analysis also suggested that known nodes downstream of Her2 were activated in response to Her2-NLP treatment. This demonstrates that Her2 protein delivery with NLPs is sufficient for the malignant transformation of nonmalignant cells. Thus, this system offers a new model for studying cell surface receptor signaling without genomic modification or transformation techniques.

Emerging Role of Exosomal Long Non-coding RNAs in Spaceflight-Associated Risks in Astronauts.

During spaceflight, astronauts are exposed to multiple unique environmental factors, particularly microgravity and ionizing radiation, that can cause a range of harmful health consequences. Over the past decades, increasing evidence demonstrates that the space environment can induce changes in gene expression and RNA processing. Long non-coding RNA (lncRNA) represent an emerging area of focus in molecular biology as they modulate chromatin structure and function, the transcription of neighboring genes, and affect RNA splicing, stability, and translation. They have been implicated in cancer development and associated with diverse cardiovascular conditions and associated risk factors. However, their role on astronauts' health after spaceflight remains poorly understood. In this perspective article, we provide new insights into the potential role of exosomal lncRNA after spaceflight. We analyzed the transcriptional profile of exosomes isolated from peripheral blood plasma of three astronauts who flew on various Shuttle missions between 1998-2001 by RNA-sequencing. Computational analysis of the transcriptome of these exosomes identified 27 differentially expressed lncRNAs with a Log2 fold change, with molecular, cellular, and clinical implications.

Single-Cell Transcriptomic Analysis of Tumor-Derived Fibroblasts and Normal Tissue-Resident Fibroblasts Reveals Fibroblast Heterogeneity in Breast Cancer.

Cancer-associated fibroblasts (CAFs) are a prominent stromal cell type in solid tumors and molecules secreted by CAFs play an important role in tumor progression and metastasis. CAFs coexist as heterogeneous populations with potentially different biological functions. Although CAFs are a major component of the breast cancer stroma, molecular and phenotypic heterogeneity of CAFs in breast cancer is poorly understood. In this study, we investigated CAF heterogeneity in triple-negative breast cancer (TNBC) using a syngeneic mouse model, BALB/c-derived 4T1 mammary tumors. Using single-cell RNA sequencing (scRNA-seq), we identified six CAF subpopulations in 4T1 tumors including: 1) myofibroblastic CAFs, enriched for α-smooth muscle actin and several other contractile proteins; 2) 'inflammatory' CAFs with elevated expression of inflammatory cytokines; and 3) a CAF subpopulation expressing major histocompatibility complex (MHC) class II proteins that are generally expressed in antigen-presenting cells. Comparison of 4T1-derived CAFs to CAFs from pancreatic cancer revealed that these three CAF subpopulations exist in both tumor types. Interestingly, cells with inflammatory and MHC class II-expressing CAF profiles were also detected in normal breast/pancreas tissue, suggesting that these phenotypes are not tumor microenvironment-induced. This work enhances our understanding of CAF heterogeneity, and specifically targeting these CAF subpopulations could be an effective therapeutic approach for treating highly aggressive TNBCs.

Comparative Molecular Analysis of Cancer Behavior Cultured In Vitro, In Vivo, and Ex Vivo.

Current pre-clinical models of cancer fail to recapitulate the cancer cell behavior in primary tumors primarily because of the lack of a deeper understanding of the effects that the microenvironment has on cancer cell phenotype. Transcriptomic profiling of 4T1 murine mammary carcinoma cells from 2D and 3D cultures, subcutaneous or orthotopic allografts (from immunocompetent or immunodeficient mice), as well as ex vivo tumoroids, revealed differences in molecular signatures including altered expression of genes involved in cell cycle progression, cell signaling and extracellular matrix remodeling. The 3D culture platforms had more in vivo-like transcriptional profiles than 2D cultures. In vivo tumors had more cells undergoing epithelial-to-mesenchymal transition (EMT) while in vitro cultures had cells residing primarily in an epithelial or mesenchymal state. Ex vivo tumoroids incorporated aspects of in vivo and in vitro culturing, retaining higher abundance of cells undergoing EMT while shifting cancer cell fate towards a more mesenchymal state. Cellular heterogeneity surveyed by scRNA-seq revealed that ex vivo tumoroids, while rapidly expanding cancer and fibroblast populations, lose a significant proportion of immune components. This study emphasizes the need to improve in vitro culture systems and preserve syngeneic-like tumor composition by maintaining similar EMT heterogeneity as well as inclusion of stromal subpopulations.

CPT1A/2-Mediated FAO Enhancement-A Metabolic Target in Radioresistant Breast Cancer.

Tumor cells, including cancer stem cells (CSCs) resistant to radio- and chemotherapy, must enhance metabolism to meet the extra energy demands to repair and survive such genotoxic conditions. However, such stress-induced adaptive metabolic alterations, especially in cancer cells that survive radiotherapy, remain unresolved. In this study, we found that CPT1 (Carnitine palmitoyl transferase I) and CPT2 (Carnitine palmitoyl transferase II), a pair of rate-limiting enzymes for mitochondrial fatty acid transportation, play a critical role in increasing fatty acid oxidation (FAO) required for the cellular fuel demands in radioresistant breast cancer cells (RBCs) and radiation-derived breast cancer stem cells (RD-BCSCs). Enhanced CPT1A/CPT2 expression was detected in the recurrent human breast cancers and associated with a worse prognosis in breast cancer patients. Blocking FAO via a FAO inhibitor or by CRISPR-mediated CPT1A/CPT2 gene deficiency inhibited radiation-induced ERK activation and aggressive growth and radioresistance of RBCs and RD-BCSCs. These results revealed that switching to FAO contributes to radiation-induced mitochondrial energy metabolism, and CPT1A/CPT2 is a potential metabolic target in cancer radiotherapy.

Single-Molecule Fluorescence Detection of the Epidermal Growth Factor Receptor in Membrane Discs.

The epidermal growth factor receptor (EGFR) is critical to normal cellular signaling pathways. Moreover, it has been implicated in a range of pathologies, including cancer. As a result, it is the primary target of many anticancer drugs. One limitation to the design and development of these drugs has been the lack of molecular-level information about the interactions and conformational dynamics of EGFR. To overcome this limitation, this work reports the construction and characterization of functional, fluorescently labeled, and full-length EGFR in model membrane nanolipoprotein particles (NLPs) for in vitro fluorescence studies. To demonstrate the utility of the system, we investigate ATP-EGFR interactions. We observe that ATP binds at the catalytic site providing a means to measure a range of distances between the catalytic site and the C-terminus via Förster resonance energy transfer (FRET). These ATP-based experiments suggest a range of conformations of the C-terminus that may be a function of the phosphorylation state for EGFR. This work is a proof-of-principle demonstration of single-molecule studies as a noncrystallographic assay for EGFR interactions in real-time and under near-physiological conditions. The diverse nature of EGFR interactions means that new tools at the molecular level have the potential to significantly enhance our understanding of receptor pathology and are of utmost importance for cancer-related drug discovery.