Flow cytometry: Potential utility in monitoring drug effects in breast cancer

Abstract Background Breast cancer bone metastasis has become one of the most common complications; however, it may cause cancer recurrence and bone nonunion, as well as local bone defects. Methods Herein, In vitro, we verified the effect of bioscaffold materials on cell proliferation and apoptosis through a CCK8 trial, staining of live/dead cells, and flow cytometry. We used immunofluorescence technology and flow cytometry to verify whether bioscaffold materials regulate macrophage polarization, and we used ALP staining, alizarin red staining and PCR to verify whether bioscaffold material promotes bone regeneration. In vivo, we once again studied the effect of bioscaffold materials on tumors by measuring tumor volume in mice, Tunel staining, and caspase-3 immunofluorescence. We also constructed a mouse skull ultimate defect model to verify the effect on bone regeneration. Results Graphene oxide (GO) nanoparticles, hydrated CePO4 nanorods and bioactive chitosan (CS) are combined to form a bioactive multifunctional CePO4/CS/GO scaffold, with characteristics such as photothermal therapy to kill tumors, macrophage polarization to promote blood vessel formation, and induction of bone formation. CePO4/CS/GO scaffold activates the caspase-3 proteasein local tumor cells, thereby lysing the DNA between nucleosomes and causing apoptosis. On the one hand, the as-released Ce3+ ions promote M2 polarization of macrophages, which secretes vascular endothelial growth factor (VEGF) and Arginase-1 (Arg-1), which promotes angiogenesis. On the other hand, the as-released Ce3+ ions also activated the BMP-2/Smad signaling pathway which facilitated bone tissue regeneration. Conclusion The multifunctional CePO4/CS/GO scaffolds may become a promising platform for therapy of breast cancer bone metastases.

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Substance P Antagonism as a Novel Therapeutic Option to Enhance Efficacy of Cisplatin in Triple Negative Breast Cancer and Protect PC12 Cells against Cisplatin-Induced Oxidative Stress and Apoptosis

Cancers ◽

10.3390/cancers13153871 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3871

Author(s):

Emma Rodriguez ◽

Guangsheng Pei ◽

Sang T. Kim ◽

Alexis German ◽

Prema Robinson

Keyword(s):

Breast Cancer ◽

Flow Cytometry ◽

Cell Line ◽

Pc12 Cells ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Therapeutic Option ◽

Neuronal Cell ◽

Receptor Antagonism ◽

Tnbc Cell

Although cisplatin is very effective as a treatment strategy in triple-negative breast cancer (TNBC), it has unwarranted outcomes owing to recurrence, chemoresistance and neurotoxicity. There is critically important to find new, effective and safe therapeutics for TNBC. We determined if SP-receptor antagonism in combination with cisplatin may serve as a novel, more efficacious and safer therapeutic option than existing therapies for TNBC. We used a neuronal cell line (PC12) and two TNBC cell lines (Sum 185 and Sum 159) for these studies. We determined that the levels of cells expressing the high-affinity SP-receptor (neurokinin 1 receptor (NK1R)), as determined by flow-cytometry was significantly elevated in response to cisplatin in all three cells. We determined that treatment with aprepitant, an SP-receptor antagonist decreased cisplatin-induced, loss of viability (studied by MTT assay), production of reactive oxygen species (by DCFDA assay) and apoptosis (by flow-cytometry) in PC12 cells while it was increased in the two TNBC cells. Furthermore, we demonstrated that important genes associated with metastases, inflammation, chemoresistance and cell cycle progression are attenuated by SP-receptor antagonism in the TNBC cell line, Sum 185. These studies implicate that SP-receptor antagonism in combination with cisplatin may possibly serve as a novel, more efficacious and safer therapeutic option than existing therapies for TNBC.

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Deciphering the temporal heterogeneity of cancer-associated fibroblast subpopulations in breast cancer

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01944-4 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Freja Albjerg Venning ◽

Kamilla Westarp Zornhagen ◽

Lena Wullkopf ◽

Jonas Sjölund ◽

Carmen Rodriguez-Cupello ◽

...

Keyword(s):

Breast Cancer ◽

Flow Cytometry ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Ex Vivo ◽

Smooth Muscle Actin ◽

Mammary Tissue ◽

Temporal Heterogeneity ◽

Alpha Smooth Muscle Actin ◽

Murine Tumours

Abstract Background Cancer-associated fibroblasts (CAFs) comprise a heterogeneous population of stromal cells within the tumour microenvironment. CAFs exhibit both tumour-promoting and tumour-suppressing functions, making them exciting targets for improving cancer treatments. Careful isolation, identification, and characterisation of CAF heterogeneity is thus necessary for ex vivo validation and future implementation of CAF-targeted strategies in cancer. Methods Murine 4T1 (metastatic) and 4T07 (poorly/non-metastatic) orthotopic triple negative breast cancer tumours were collected after 7, 14, or 21 days. The tumours were analysed via flow cytometry for the simultaneous expression of six CAF markers: alpha smooth muscle actin (αSMA), fibroblast activation protein alpha (FAPα), platelet derived growth factor receptor alpha and beta (PDGFRα and PDGFRβ), CD26/DPP4 and podoplanin (PDPN). All non-CAFs were excluded from the analysis using a lineage marker cocktail (CD24, CD31, CD45, CD49f, EpCAM, LYVE-1, and TER-119). In total 128 murine tumours and 12 healthy mammary fat pads were analysed. Results We have developed a multicolour flow cytometry strategy based on exclusion of non-CAFs and successfully employed this to explore the temporal heterogeneity of freshly isolated CAFs in the 4T1 and 4T07 mouse models of triple-negative breast cancer. Analysing 128 murine tumours, we identified 5–6 main CAF populations and numerous minor ones based on the analysis of αSMA, FAPα, PDGFRα, PDGFRβ, CD26, and PDPN. All markers showed temporal changes with a distinct switch from primarily PDGFRα+ fibroblasts in healthy mammary tissue to predominantly PDGFRβ+ CAFs in tumours. CD26+ CAFs emerged as a large novel subpopulation, only matched by FAPα+ CAFs in abundance. Conclusion We demonstrate that multiple subpopulations of CAFs co-exist in murine triple negative breast cancer, and that the abundance and dynamics for each marker differ depending on tumour type and time. Our results form the foundation needed to isolate and characterise specific CAF populations, and ultimately provide an opportunity to therapeutically target specific CAF subpopulations.

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