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.

Clinical Outcomes Using Magnetic Seeds as a Non-wire, Non-radioactive Alternative for Localization of Non-palpable Breast Lesions.

BACKGROUND: Nonpalpable breast lesions require precise preoperative localization to facilitate negative margins with breast-conserving therapy. The traditional use of wires has several challenges including patient discomfort, wire migration, and coordination of schedules between radiology and the operating room. Radioactive seed localization overcomes some of these challenges, but radiation safety requirements have limited adoption of this technology. The authors examined their institutional experience with Magseed as an alternative technology for localization and compared outcomes with those of wire and radioactive seed localization. METHODS: An institutional review board (IRB)-approved retrospective study was performed to evaluate patients who underwent excisional biopsy or segmental mastectomy after wire-guided localization (WGL), radioactive seed localization (RSL), or Magseed localization (ML). The clinical and pathologic factors of the three groups were assessed with a negative margin rate as the primary outcome measure. RESULTS: Of the 1835 patients in the study, 825 underwent WGL, 449 underwent RSL, and 561 underwent ML. For the patients with either multiple lesions or a large lesion that required bracketing, multiple localization devices were placed in 31% of the WGL patients, 28% of the RSL patients, and 23% of the ML patients (p = 0.006). Negative margins were achieved in 91% of the WGL patients, 89% of the RSL patients, and 89% of the ML patients (p = 0.4). CONCLUSION: Localization of non-palpable breast lesions using Magseed is a safe and effective alternative to WGL and RSL that overcomes radiation safety limitations and increases radiology and surgery scheduling efficiency.

Oncogenic KRAS Reduces Expression of FGF21 in Acinar Cells to Promote Pancreatic Tumorigenesis in Mice on a High-Fat Diet.

BACKGROUND & AIMS: Obesity is a risk factor for pancreatic cancer. In mice, a high-fat diet (HFD) and expression of oncogenic KRAS lead to development of invasive pancreatic ductal adenocarcinoma (PDAC) by unknown mechanisms. We investigated how oncogenic KRAS regulates the expression of fibroblast growth factor 21, FGF21, a metabolic regulator that prevents obesity, and the effects of recombinant human FGF21 (rhFGF21) on pancreatic tumorigenesis. METHODS: We performed immunohistochemical analyses of FGF21 levels in human pancreatic tissue arrays, comprising 59 PDAC specimens and 45 nontumor tissues. We also studied mice with tamoxifen-inducible expression of oncogenic KRAS in acinar cells (KrasG12D/+ mice) and fElasCreERT mice (controls). KrasG12D/+ mice were placed on an HFD or regular chow diet (control) and given injections of rhFGF21 or vehicle; pancreata were collected and analyzed by histology, immunoblots, quantitative polymerase chain reaction, and immunohistochemistry. We measured markers of inflammation in the pancreas, liver, and adipose tissue. Activity of RAS was measured based on the amount of bound guanosine triphosphate. RESULTS: Pancreatic tissues of mice expressed high levels of FGF21 compared with liver tissues. FGF21 and its receptor proteins were expressed by acinar cells. Acinar cells that expressed KrasG12D/+ had significantly lower expression of Fgf21 messenger RNA compared with acinar cells from control mice, partly due to down-regulation of PPARG expression-a transcription factor that activates Fgf21 transcription. Pancreata from KrasG12D/+ mice on a control diet and given injections of rhFGF21 had reduced pancreatic inflammation, infiltration by immune cells, and acinar-to-ductal metaplasia compared with mice given injections of vehicle. HFD-fed KrasG12D/+ mice given injections of vehicle accumulated abdominal fat, developed extensive inflammation, pancreatic cysts, and high-grade pancreatic intraepithelial neoplasias (PanINs); half the mice developed PDAC with liver metastases. HFD-fed KrasG12D/+ mice given injections of rhFGF21 had reduced accumulation of abdominal fat and pancreatic triglycerides, fewer pancreatic cysts, reduced systemic and pancreatic markers of inflammation, fewer PanINs, and longer survival-only approximately 12% of the mice developed PDACs, and none of the mice had metastases. Pancreata from HFD-fed KrasG12D/+ mice given injections of rhFGF21 had lower levels of active RAS than from mice given vehicle. CONCLUSIONS: Normal acinar cells from mice and humans express high levels of FGF21. In mice, acinar expression of oncogenic KRAS significantly reduces FGF21 expression. When these mice are placed on an HFD, they develop extensive inflammation, pancreatic cysts, PanINs, and PDACs, which are reduced by injection of FGF21. FGF21 also reduces the guanosine triphosphate binding capacity of RAS. FGF21 might be used in the prevention or treatment of pancreatic cancer.

Alkaline ceramidase 2 is essential for the homeostasis of plasma sphingoid bases and their phosphates.

Sphingosine-1-phosphate (S1P) plays important roles in cardiovascular development and immunity. S1P is abundant in plasma because erythrocytes-the major source of S1P-lack any S1P-degrading activity; however, much remains unclear about the source of the plasma S1P precursor, sphingosine (SPH), derived mainly from the hydrolysis of ceramides by the action of ceramidases that are encoded by 5 distinct genes, acid ceramidase 1 ( ASAH1)/ Asah1, ASAH2/ Asah2, alkaline ceramidase 1 ( ACER1)/ Acer1, ACER2/ Acer2, and ACER3/ Acer3, in humans/mice. Previous studies have reported that knocking out Asah1 or Asah2 failed to reduce plasma SPH and S1P levels in mice. In this study, we show that knocking out Acer1 or Acer3 also failed to reduce the blood levels of SPH or S1P in mice. In contrast, knocking out Acer2 from either whole-body or the hematopoietic lineage markedly decreased the blood levels of SPH and S1P in mice. Of interest, knocking out Acer2 from whole-body or the hematopoietic lineage also markedly decreased the levels of dihydrosphingosine (dhSPH) and dihydrosphingosine-1-phosphate (dhS1P) in blood. Taken together, these results suggest that ACER2 plays a key role in the maintenance of high plasma levels of sphingoid base-1-phosphates-S1P and dhS1P-by controlling the generation of sphingoid bases-SPH and dhSPH-in hematopoietic cells.-Li, F., Xu, R., Low, B. E., Lin, C.-L., Garcia-Barros, M., Schrandt, J., Mileva, I., Snider, A., Luo, C. K., Jiang, X.-C., Li, M.-S., Hannun, Y. A., Obeid, L. M., Wiles, M. V., Mao, C. Alkaline ceramidase 2 is essential for the homeostasis of plasma sphingoid bases and their phosphates.

Positive Quantitative Relationship between EMT and Contact-Initiated Sliding on Fiber-like Tracks.

Epithelial-mesenchymal transition (EMT) is a complex process by which cells acquire invasive properties that enable escape from the primary tumor. Complete EMT, however, is not required for metastasis: circulating tumor cells exhibit hybrid epithelial-mesenchymal states, and genetic perturbations promoting partial EMT induce metastasis in vivo. An open question is whether and to what extent intermediate stages of EMT promote invasiveness. Here, we investigate this question, building on recent observation of a new invasive property. Migrating cancer cell lines and cells transduced with prometastatic genes slide around other cells on spatially confined, fiberlike micropatterns. We show here that low-dosage/short-duration exposure to transforming growth factor beta (TGFß) induces partial EMT and enables sliding on narrower (26 µm) micropatterns than untreated counterparts (41 µm). High-dosage/long-duration exposure induces more complete EMT, including disrupted cell-cell contacts and reduced E-cadherin expression, and promotes sliding on the narrowest (15 µm) micropatterns. These results identify a direct and quantitative relationship between EMT and cell sliding and show that EMT-associated invasive sliding is progressive, with cells that undergo partial EMT exhibiting intermediate sliding behavior and cells that transition more completely through EMT displaying maximal sliding. Our findings suggest a model in which fiber maturation and EMT work synergistically to promote invasiveness during cancer progression.

Social Determinants in COVID-19 Experiences of Children With Disabilities Receiving School-Based Services in Chicago: Mixed-Methods Study of Parent/Guardian Perspectives.

OBJECTIVE: To identify the impact of social determinants on the experiences of children with disabilities and their families during the COVID-19 pandemic from the perspective of parents/guardians. METHODS: A mixed-methods study engaged parents/guardians of children with Individualized Education Programs (IEPs) in July to August 2021 at a developmental/behavioral pediatrics clinic in 1 urban academic medical center. All parents/guardians completed study-specific surveys on experiences and impact of COVID-19. A subset completed semi-structured interviews. Analysis included descriptive statistics and Fisher exact tests for survey questions and thematic analysis to code interviews and identify themes. Results were corroborated by experts in developmental/behavioral pediatrics and special education. RESULTS: Participants included 24 parents/guardians representing 27 children (mean = 7.37 years). A majority attended public school (78%) and identified as non-White (78%). Most commonly, the children's disabilities were autism (52%), attention-deficit hyperactivity disorder (37%), and speech/language impairment (33%). The services received by children most commonly were speech/language (89%) and physical/occupational (70%) therapies. Five themes emerged about the impact of social determinants on experiences during COVID-19 related to: adapting to disruption of routines, attendance/engagement in learning, interruption of IEP services, support for children and families, and challenges with technology. CONCLUSION: Social determinants, such as housing, income, insurance, and quality of education, affected the experiences of families and their ability to adapt to the needs of children with disabilities in the setting of COVID-19 pandemic-related changes.

Selective killing of cancer cells harboring mutant RAS by concomitant inhibition of NADPH oxidase and glutathione biosynthesis.

Oncogenic RAS is a critical driver for the initiation and progression of several types of cancers. However, effective therapeutic strategies by targeting RAS, in particular RASG12D and RASG12V, and associated downstream pathways have been so far unsuccessful. Treatment of oncogenic RAS-ravaged cancer patients remains a currently unmet clinical need. Consistent with a major role in cancer metabolism, oncogenic RAS activation elevates both reactive oxygen species (ROS)-generating NADPH oxidase (NOX) activity and ROS-scavenging glutathione biosynthesis. At a certain threshold, the heightened oxidative stress and antioxidant capability achieve a higher level of redox balance, on which cancer cells depend to gain a selective advantage on survival and proliferation. However, this prominent metabolic feature may irrevocably render cancer cells vulnerable to concurrent inhibition of both NOX activity and glutathione biosynthesis, which may be exploited as a novel therapeutic strategy. In this report, we test this hypothesis by treating the HRASG12V-transformed ovarian epithelial cells, mutant KRAS-harboring pancreatic and colon cancer cells of mouse and human origins, as well as cancer xenografts, with diphenyleneiodonium (DPI) and buthionine sulfoximine (BSO) combination, which inhibit NOX activity and glutathione biosynthesis, respectively. Our results demonstrate that concomitant targeting of NOX and glutathione biosynthesis induces a highly potent lethality to cancer cells harboring oncogenic RAS. Therefore, our studies provide a novel strategy against RAS-bearing cancers that warrants further mechanistic and translational investigation.

The Glutaminase Inhibitor CB-839 (Telaglenastat) Enhances the Antimelanoma Activity of T-Cell-Mediated Immunotherapies.

Immune-checkpoint inhibitors and adoptive tumor-infiltrating lymphocyte (TIL) therapies have profoundly improved the survival of patients with melanoma. However, a majority of patients do not respond to these agents, and many responders experience disease relapse. Although numerous innovative treatments are being explored to offset the limitations of these agents, novel therapeutic combinations with immunotherapies have the potential to improve patient responses. In this study, we evaluated the antimelanoma activity of immunotherapy combinations with Telaglenastat (CB-839), a potent glutaminase inhibitor (GLSi) that has favorable systemic tolerance. In in vitro TIL:tumor coculture studies, CB-839 treatment improved the cytotoxic activity of autologous TILs on patient-derived melanoma cells. CB-839 treatment decreased the conversion of glutamine to alpha-ketoglutarate (αKGA) more potently in tumor cells versus TILs in these cocultures. These results suggest that CB-839 may improve immune function in a tumor microenvironment by differentially altering tumor and immune cell metabolism. In vivo CB-839 treatment activated melanoma antigen-specific T cells and improved their tumor killing activity in an immune-competent mouse model of adoptive T-cell therapy. Additionally, the combination of CB-839 with anti-PD1 or anti-CTLA4 antibodies increased tumor infiltration by effector T cells and improved the antitumor activity of these checkpoint inhibitors in a high mutation burden mouse melanoma model. Responsiveness to these treatments was also accompanied by an increase of interferon gamma (IFNγ)-associated gene expression in the tumors. Together, these results provide a strong rationale for combining CB-839 with immune therapies to improve efficacy of these treatments against melanoma.

Multivariate relationships among nucleus and Golgi properties during fibrillar migration are robust to and unchanged by epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT) and maturation of a fibrillar tumor microenvironment play important roles in breast cancer progression. A better understanding of how these events promote cancer cell migration and invasion could help identify new strategies to curb metastasis. The nucleus and Golgi affect migration in a microenvironment-dependent manner. Nucleus size and mechanics influence the ability of a cell to squeeze through confined tumor microenvironments. Golgi positioning determines front-rear polarity necessary for migration. While the roles of individual attributes of nucleus and Golgi in migration are being clarified, how their manifold features are inter-related and work together remains to be understood at a systems level. Here, to elucidate relationships among nucleus and Golgi properties, we quantified twelve morphological and positional properties of these organelles during fibrillar migration of human mammary epithelial cells. Principal component analysis (PCA) reduced the twelve-dimensional space of measured properties to three principal components that capture 75% of the variations in organelle features. Unexpectedly, nucleus and Golgi properties that co-varied in a PCA model built with data from untreated cells were largely similar to co-variations identified using data from TGFß-treated cells. Thus, while TGFß-mediated EMT significantly alters gene expression and motile phenotype, it did not significantly affect the relationships among nucleus size, aspect ratio and orientation with migration direction and among Golgi size and nucleus-Golgi separation distance. Indeed, in a combined PCA model incorporating data from untreated and TGFß-treated cells, scores of individual cells occupy overlapping regions in principal component space, indicating that TGFß-mediated EMT does not promote a unique "Golgi-nucleus phenotype" during fibrillar migration. These results suggest that migration along spatially-confined fiber-like tracks employs a conserved nucleus-Golgi arrangement that is independent of EMT state.