Asbestos exposure determined 357 days after death through autopsy: a report of a multidisciplinary approach.

Asbestosis is an interstitial lung disease caused by the inhalation of asbestos fibers and poses a significant risk to individuals working in construction, shipping, mining, and related industries. In a forensic context, postmortem investigations are crucial for accurate diagnosis, for which the gold standard is the histopathological examination. This case report describes the autopsy and related investigations conducted on an 84-year-old man, nearly one year (357 days) after his death. After a post-mortem CT scan, an autoptic investigation was performed, followed by histopathological, immunohistochemical, and scanning electron microscopy examinations. The integration of the evidence from these examinations with previously available personal and clinical information conclusively confirmed the diagnosis of asbestosis. We demonstrated the efficacy and reliability of our diagnostic protocol in detecting asbestosis and asbestos fibers and excluding mesothelioma even in decomposed tissues. According to our findings autopsy remains the diagnostic gold standard in cases of suspected asbestosis within a forensic context, even 1 year after death, therefore it is always highly recommended, even in cases where the body has decomposed.

PI3K-driven HER2 expression is a potential therapeutic target in colorectal cancer stem cells.

OBJECTIVE: Cancer stem cells are responsible for tumour spreading and relapse. Human epidermal growth factor receptor 2 (HER2) expression is a negative prognostic factor in colorectal cancer (CRC) and a potential target in tumours carrying the gene amplification. Our aim was to define the expression of HER2 in colorectal cancer stem cells (CR-CSCs) and its possible role as therapeutic target in CRC resistant to anti- epidermal growth factor receptor (EGFR) therapy. DESIGN: A collection of primary sphere cell cultures obtained from 60 CRC specimens was used to generate CR-CSC mouse avatars to preclinically validate therapeutic options. We also made use of the ChIP-seq analysis for transcriptional evaluation of HER2 activation and global RNA-seq to identify the mechanisms underlying therapy resistance. RESULTS: Here we show that in CD44v6-positive CR-CSCs, high HER2 expression levels are associated with an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which promotes the acetylation at the regulatory elements of the Erbb2 gene. HER2 targeting in combination with phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MEK) inhibitors induces CR-CSC death and regression of tumour xenografts, including those carrying Kras and Pik3ca mutation. Requirement for the triple targeting is due to the presence of cancer-associated fibroblasts, which release cytokines able to confer CR-CSC resistance to PI3K/AKT inhibitors. In contrast, targeting of PI3K/AKT as monotherapy is sufficient to kill liver-disseminating CR-CSCs in a model of adjuvant therapy. CONCLUSIONS: While PI3K targeting kills liver-colonising CR-CSCs, the concomitant inhibition of PI3K, HER2 and MEK is required to induce regression of tumours resistant to anti-EGFR therapies. These data may provide a rationale for designing clinical trials in the adjuvant and metastatic setting.

Cancer-associated fibroblasts as abettors of tumor progression at the crossroads of EMT and therapy resistance.

In the last decades, the role of the microenvironment in tumor progression and therapeutic outcome has gained increasing attention. Cancer-associated fibroblasts (CAFs) have emerged as key players among stromal cells, owing to their abundance in most solid tumors and their diverse tumor-restraining/promoting roles. The interplay between tumor cells and neighboring CAFs takes place by both paracrine signals (cytokines, exosomes and metabolites) or by the multifaceted functions of the surrounding extracellular matrix. Here, we dissect the most recent identified mechanisms underlying CAF-mediated control of tumor progression and therapy resistance, which include induction of the epithelial-to-mesenchymal transition (EMT), activation of survival pathways or stemness-related programs and metabolic reprogramming in tumor cells. Importantly, the recently unveiled heterogeneity in CAFs claims tailored therapeutic efforts aimed at eradicating the specific subset facilitating tumor progression, therapy resistance and relapse. However, despite the large amount of pre-clinical data, much effort is still needed to translate CAF-directed anti-cancer strategies from the bench to the clinic.

CHK1-targeted therapy to deplete DNA replication-stressed, p53-deficient, hyperdiploid colorectal cancer stem cells.

OBJECTIVE: Cancer stem cells (CSCs) are responsible for tumour formation and spreading, and their targeting is required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer (CRC), particularly for tumours carrying RAS-activating mutations. The aim of this study was to identify novel CSC-targeting strategies. DESIGN: To discover potential therapeutics to be clinically investigated as single agent, we performed a screening with a panel of FDA-approved or investigational drugs on primary CRC cells enriched for CSCs (CRC-SCs) isolated from 27 patients. Candidate predictive biomarkers of efficacy were identified by integrating genomic, reverse-phase protein microarray (RPPA) and cytogenetic analyses, and validated by immunostainings. DNA replication stress (RS) was increased by employing DNA replication-perturbing or polyploidising agents. RESULTS: The drug-library screening led to the identification of LY2606368 as a potent anti-CSC agent acting in vitro and in vivo in tumour cells from a considerable number of patients (∼36%). By inhibiting checkpoint kinase (CHK)1, LY2606368 affected DNA replication in most CRC-SCs, including RAS-mutated ones, forcing them into premature, lethal mitoses. Parallel genomic, RPPA and cytogenetic analyses indicated that CRC-SCs sensitive to LY2606368 displayed signs of ongoing RS response, including the phosphorylation of RPA32 and ataxia telangiectasia mutated serine/threonine kinase (ATM). This was associated with mutation(s) in TP53 and hyperdiploidy, and made these CRC-SCs exquisitely dependent on CHK1 function. Accordingly, experimental increase of RS sensitised resistant CRC-SCs to LY2606368. CONCLUSIONS: LY2606368 selectively eliminates replication-stressed, p53-deficient and hyperdiploid CRC-SCs independently of RAS mutational status. These results provide a strong rationale for biomarker-driven clinical trials with LY2606368 in patients with CRC.

Lipid droplets: a new player in colorectal cancer stem cells unveiled by spectroscopic imaging.

The cancer stem cell (CSC) model is describing tumors as a hierarchical organized system and CSCs are suggested to be responsible for cancer recurrence after therapy. The identification of specific markers of CSCs is therefore of paramount importance. Here, we show that high levels of lipid droplets (LDs) are a distinctive mark of CSCs in colorectal (CR) cancer. This increased lipid content was clearly revealed by label-free Raman spectroscopy and it directly correlates with well-accepted CR-CSC markers as CD133 and Wnt pathway activity. By xenotransplantation experiments, we have finally demonstrated that CR-CSCs overexpressing LDs retain most tumorigenic potential. A relevant conceptual advance in this work is the demonstration that a cellular organelle, the LD, is a signature of CSCs, in addition to molecular markers. A further functional characterization of LDs could lead soon to design new target therapies against CR-CSCs.

Human NK cells selective targeting of colon cancer-initiating cells: a role for natural cytotoxicity receptors and MHC class I molecules.

Tumor cell populations have been recently proposed to be composed of two compartments: tumor-initiating cells characterized by a slow and asymmetrical growth, and the "differentiated" cancer cells with a fast and symmetrical growth. Cancer stem cells or cancer-initiating cells (CICs) play a crucial role in tumor recurrence. The resistance of CICs to drugs and irradiation often allows them to survive traditional therapy. NK cells are potent cytotoxic lymphocytes that can recognize tumor cells. In this study, we have analyzed the NK cell recognition of tumor target cells derived from the two cancer cell compartments of colon adenocarcinoma lesions. Our data demonstrate that freshly purified allogeneic NK cells can recognize and kill colorectal carcinoma-derived CICs whereas the non-CIC counterpart of the tumors (differentiated tumor cells), either autologous or allogeneic, is less susceptible to NK cells. This difference in the NK cell susceptibility correlates with higher expression on CICs of ligands for NKp30 and NKp44 in the natural cytotoxicity receptor (NCR) group of activating NK receptors. In contrast, CICs express lower levels of MHC class I, known to inhibit NK recognition, on their surface than do the "differentiated" tumor cells. These data have been validated by confocal microscopy where NCR ligands and MHC class I molecule membrane distribution have been analyzed. Moreover, NK cell receptor blockade in cytotoxicity assays demonstrates that NCRs play a major role in the recognition of CIC targets. This study strengthens the idea that biology-based therapy harnessing NK cells could be an attractive opportunity in solid tumors.

Tumor and its microenvironment: a synergistic interplay.

The mutual and interdependent interaction between tumor and its microenvironment is a crucial topic in cancer research. Recently, it was reported that targeting stromal events could improve efficacies of current therapeutics and prevent metastatic spreading. Tumor microenvironment is a "complex network" of different cell types, soluble factors, signaling molecules and extracellular matrix components, which orchestrate the fate of tumor progression. As by definition, cancer stem cells (CSCs) are proposed to be the unique cell type able to maintain tumor mass and survive outside the primary tumor at metastatic sites. Being exposed to environmental stressors, including reactive oxygen species (ROS), CSCs have developed a GSH-dependent antioxidant system to improve ROS defense capability and acquire a malignant phenotype. Nevertheless, tumor progression is dependent on extracellular matrix remodeling, fibroblasts and macrophages activation in response to oxidative stress, as well as epithelial mesenchymal transition (EMT)-inducing signals and endothelial and perivascular cells recruitment. Besides providing a survival advantage by inducing de novo angiogenesis, tumor-associated vessels contribute to successful dissemination by facilitating tumor cells entry into the circulatory system and driving the formation of pre-metastatic niche. In this review, we focus on the synergistic effect of hypoxia inducible factors (HIFs) and vascular endothelial growth factors (VEGFs) in the successful outgrowth of metastasis, integrating therefore many of the emerging models and theories in the field.

The miRNA Contribution in Adipocyte Maturation.

Mesenchymal stem cells, due to their multipotent ability, are considered one of the best candidates to be used in regenerative medicine. To date, the most used source is represented by the bone marrow, despite the limited number of cells and the painful/invasive procedure for collection. Therefore, the scientific community has investigated many alternative sources for the collection of mesenchymal stem cells, with the adipose tissue representing the best option, given the abundance of mesenchymal stem cells and the easy access. Although adipose mesenchymal stem cells have recently been investigated for their multipotency, the molecular mechanisms underlying their adipogenic potential are still unclear. In this scenario, this communication is aimed at defining the role of miRNAs in adipogenic potential of adipose-derived mesenchymal stem cells via real-time PCR. Even if preliminary, our data show that cell culture conditions affect the expression of specific miRNA involved in the adipogenic potential of mesenchymal stem cells. The in vitro/in vivo validation of these results could pave the way for novel therapeutic strategies in the field of regenerative medicine. In conclusion, our research highlights how specific cell culture conditions can modulate the adipogenic potential of adipose mesenchymal stem cells through the regulation of specific miRNAs.

Proliferation state and polo-like kinase1 dependence of tumorigenic colon cancer cells.

Tumor-initiating cells are responsible for tumor maintenance and relapse in solid and hematologic cancers. Although tumor-initiating cells were initially believed to be mainly quiescent, rapidly proliferating tumorigenic cells were found in breast cancer. In colon cancer, the proliferative activity of the tumorigenic population has not been defined, although it represents an essential parameter for the development of more effective therapeutic strategies. Here, we show that tumorigenic colon cancer cells can be found in a rapidly proliferating state in vitro and in vivo, both in human tumors and mouse xenografts. Inhibitors of polo-like kinase1 (Plk1), a mitotic kinase essential for cell proliferation, demonstrated maximal efficiency over other targeted compounds and chemotherapeutic agents in inducing death of colon cancer-initiating cells in vitro. In vivo, Plk1 inhibitors killed CD133(+) colon cancer cells leading to complete growth arrest of colon cancer stem cell-derived xenografts, whereas chemotherapeutic agents only slowed tumor progression. While chemotherapy treatment increased CD133(+) cell proliferation, treatment with Plk1 inhibitors eliminated all proliferating tumor-initiating cells. Quiescent CD133(+) cells that survived the treatment with Plk1 inhibitors could be killed by subsequent Plk1 inhibition when they exited from quiescence. Altogether, these results provide a new insight into the proliferative status of colon tumor-initiating cells both in basal conditions and in response to therapy and indicate Plk1 inhibitors as potentially useful in the treatment of colorectal cancer.

Subtype-specific kinase dependency regulates growth and metastasis of poor-prognosis mesenchymal colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) can be divided into four consensus molecular subtypes (CMS), each with distinct biological features. CMS4 is associated with epithelial-mesenchymal transition and stromal infiltration (Guinney et al., Nat Med 21:1350-6, 2015; Linnekamp et al., Cell Death Differ 25:616-33, 2018), whereas clinically it is characterized by lower responses to adjuvant therapy, higher incidence of metastatic spreading and hence dismal prognosis (Buikhuisen et al., Oncogenesis 9:66, 2020). METHODS: To understand the biology of the mesenchymal subtype and unveil specific vulnerabilities, a large CRISPR-Cas9 drop-out screen was performed on 14 subtyped CRC cell lines to uncover essential kinases in all CMSs. Dependency of CMS4 cells on p21-activated kinase 2 (PAK2) was validated in independent 2D and 3D in vitro cultures and in vivo models assessing primary and metastatic outgrowth in liver and peritoneum. TIRF microscopy was used to uncover actin cytoskeleton dynamics and focal adhesion localization upon PAK2 loss. Subsequent functional assays were performed to determine altered growth and invasion patterns. RESULTS: PAK2 was identified as a key kinase uniquely required for growth of the mesenchymal subtype CMS4, both in vitro and in vivo. PAK2 plays an important role in cellular attachment and cytoskeletal rearrangements (Coniglio et al., Mol Cell Biol 28:4162-72, 2008; Grebenova et al., Sci Rep 9:17171, 2019). In agreement, deletion or inhibition of PAK2 impaired actin cytoskeleton dynamics in CMS4 cells and, as a consequence, significantly reduced invasive capacity, while it was dispensable for CMS2 cells. Clinical relevance of these findings was supported by the observation that deletion of PAK2 from CMS4 cells prevented metastatic spreading in vivo. Moreover, growth in a model for peritoneal metastasis was hampered when CMS4 tumor cells were deficient for PAK2. CONCLUSION: Our data reveal a unique dependency of mesenchymal CRC and provide a rationale for PAK2 inhibition to target this aggressive subgroup of colorectal cancer.

Recapitulating thyroid cancer histotypes through engineering embryonic stem cells.

Thyroid carcinoma (TC) is the most common malignancy of endocrine organs. The cell subpopulation in the lineage hierarchy that serves as cell of origin for the different TC histotypes is unknown. Human embryonic stem cells (hESCs) with appropriate in vitro stimulation undergo sequential differentiation into thyroid progenitor cells (TPCs-day 22), which maturate into thyrocytes (day 30). Here, we create follicular cell-derived TCs of all the different histotypes based on specific genomic alterations delivered by CRISPR-Cas9 in hESC-derived TPCs. Specifically, TPCs harboring BRAFV600E or NRASQ61R mutations generate papillary or follicular TC, respectively, whereas addition of TP53R248Q generate undifferentiated TCs. Of note, TCs arise by engineering TPCs, whereas mature thyrocytes have a very limited tumorigenic capacity. The same mutations result in teratocarcinomas when delivered in early differentiating hESCs. Tissue Inhibitor of Metalloproteinase 1 (TIMP1)/Matrix metallopeptidase 9 (MMP9)/Cluster of differentiation 44 (CD44) ternary complex, in cooperation with Kisspeptin receptor (KISS1R), is involved in TC initiation and progression. Increasing radioiodine uptake, KISS1R and TIMP1 targeting may represent a therapeutic adjuvant option for undifferentiated TCs.

BCL-XL inhibition induces an FGFR4-mediated rescue response in colorectal cancer.

The heterogeneous therapy response observed in colorectal cancer is in part due to cancer stem cells (CSCs) that resist chemotherapeutic insults. The anti-apoptotic protein BCL-XL plays a critical role in protecting CSCs from cell death, where its inhibition with high doses of BH3 mimetics can induce apoptosis. Here, we screen a compound library for synergy with low-dose BCL-XL inhibitor A-1155463 to identify pathways that regulate sensitivity to BCL-XL inhibition and reveal that fibroblast growth factor receptor (FGFR)4 inhibition effectively sensitizes to A-1155463 both in vitro and in vivo. Mechanistically, we identify a rescue response that is activated upon BCL-XL inhibition and leads to rapid FGF2 secretion and subsequent FGFR4-mediated post-translational stabilization of MCL-1. FGFR4 inhibition prevents MCL-1 upregulation and thereby sensitizes CSCs to BCL-XL inhibition. Altogether, our findings suggest a cell transferable induction of a FGF2/FGFR4 rescue response in CRC that is induced upon BCL-XL inhibition and leads to MCL-1 upregulation.

Targeting of the Peritumoral Adipose Tissue Microenvironment as an Innovative Antitumor Therapeutic Strategy.

The tumor microenvironment (TME) plays a key role in promoting and sustaining cancer growth. Adipose tissue (AT), due to its anatomical distribution, is a prevalent component of TME, and contributes to cancer development and progression. Cancer-associated adipocytes (CAAs), reprogrammed by cancer stem cells (CSCs), drive cancer progression by releasing metabolites and inflammatory adipokines. In this review, we highlight the mechanisms underlying the bidirectional crosstalk among CAAs, CSCs, and stromal cells. Moreover, we focus on the recent advances in the therapeutic targeting of adipocyte-released factors as an innovative strategy to counteract cancer progression.

Destroying the Shield of Cancer Stem Cells: Natural Compounds as Promising Players in Cancer Therapy.

In a scenario where eco-sustainability and a reduction in chemotherapeutic drug waste are certainly a prerogative to safeguard the biosphere, the use of natural products (NPs) represents an alternative therapeutic approach to counteract cancer diseases. The presence of a heterogeneous cancer stem cell (CSC) population within a tumor bulk is related to disease recurrence and therapy resistance. For this reason, CSC targeting presents a promising strategy for hampering cancer recurrence. Increasing evidence shows that NPs can inhibit crucial signaling pathways involved in the maintenance of CSC stemness and sensitize CSCs to standard chemotherapeutic treatments. Moreover, their limited toxicity and low costs for large-scale production could accelerate the use of NPs in clinical settings. In this review, we will summarize the most relevant studies regarding the effects of NPs derived from major natural sources, e.g., food, botanical, and marine species, on CSCs, elucidating their use in pre-clinical and clinical studies.

Correlative Raman-Electron-Light (CREL) Microscopy Analysis of Lipid Droplets in Melanoma Cancer Stem Cells.

Among all neoplasms, melanoma is characterized by a very high percentage of cancer stem cells (CSCs). Several markers have been proposed for their identification, and lipid droplets (LDs) are among them. Different techniques are used for their characterization such as mass spectrometry, imaging techniques, and vibrational spectroscopies. Some emerging experimental approaches for the study of LDs are represented by correlative light-electron microscopy and by correlative Raman imaging-scanning electron microscopy (SEM). Based on these scientific approaches, we developed a novel methodology (CREL) by combining Raman micro-spectroscopy, confocal fluorescence microscopy, and SEM coupled with an energy-dispersive X-ray spectroscopy module. This procedure correlated cellular morphology, chemical properties, and spatial distribution from the same region of interest, and in this work, we presented the application of CREL for the analysis of LDs within patient-derived melanoma CSCs (MCSCs).

Dual Inhibition of Myc Transcription and PI3K Activity Effectively Targets Colorectal Cancer Stem Cells.

Despite advances in the curative approach, the survival rate of advanced colorectal cancer (CRC) patients is still poor, which is likely due to the emergence of cancer cell clones resistant to the available therapeutic options. We have already shown that CD44v6-positive CRC stem cells (CR-CSCs) are refractory toward standard anti-tumor therapeutic agents due to the activation of the PI3K pathway together with high HER2 expression levels. Tumor microenvironmental cytokines confer resistance to CR-CSCs against HER2/PI3K targeting by enhancing activation of the MAPK pathway. Here, we show that the CSC compartment, spared by BRAF inhibitor-based targeted therapy, is associated with increased expression levels of CD44v6 and Myc and retains boosted clonogenic activity along with residual tumorigenic potential. Inhibition of Myc transcription, downstream of the MAPK cascade components, and PI3K pathway activity was able to overcome the protective effects of microenvironmental cytokines, affecting the survival and the clonogenic activity of CR-CSCs, regardless of their mutational background. Likewise, the double targeting induced stabilization of mouse tumor avatars. Altogether, these data outline the rationale for dual kinase targeting of CR-CSCs to prevent their adaptive response, which would lead to disease progression.

Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51.

Breast cancer (BC) is the second cause of cancer-related deceases in the worldwide female population. Despite the successful treatment advances, 25% of BC develops resistance to current therapeutic regimens, thereby remaining a major hurdle for patient management. Current therapies, targeting the molecular events underpinning the adaptive resistance, still require effort to improve BC treatment. Using BC sphere cells (BCSphCs) as a model, here we showed that BC stem-like cells express high levels of Myc, which requires the presence of the multifunctional DNA/RNA binding protein Sam68 for the DNA-damage repair. Analysis of a cohort of BC patients displayed that Sam68 is an independent negative factor correlated with the progression of the disease. Genetic inhibition of Sam68 caused a defect in PARP-induced PAR chain synthesis upon DNA-damaging insults, resulting in cell death of TNBC cells. In contrast, BC stem-like cells were able to survive due to an upregulation of Rad51. Importantly, the inhibition of Rad51 showed synthetic lethal effect with the silencing of Sam68, hampering the cell viability of patient-derived BCSphCs and stabilizing the growth of tumor xenografts, including those TNBC carrying BRCA mutation. Moreover, the analysis of Myc, Sam68 and Rad51 expression demarcated a signature of a poor outcome in a large cohort of BC patients. Thus, our findings suggest the importance of targeting Sam68-PARP1 axis and Rad51 as potential therapeutic candidates to counteract the expansion of BC cells with an aggressive phenotype.

Adipose stromal cells promote the transition of colorectal cancer cells toward a mesenchymal-like phenotype.

Colon cancer progression is among the risks that increase with obesity. We have recently unveiled the molecular mechanism by which adipose tissue-released molecules, HGF and IL-6, make colorectal cancer (CRC) cells acquiring mesenchymal traits. Targeting of adipose-derived factors abrogate the metastatic potential of CRC stem cells (CR-CSCs) in obese patients.

FACS-based protocol to assess cytotoxicity and clonogenic potential of colorectal cancer stem cells using a Wnt/ß-catenin signaling pathway reporter.

Cancer stem cells (CSCs) play a key role in tumor initiation and progression. A real-time tool to evaluate the activation of CSC-specific signaling pathways is crucial for the study of this cancer cell subset. Here, we present a protocol to monitor, in vitro, the activation of Wnt/ß-catenin signaling pathway, which is considered a functional biomarker for colorectal CSCs (CR-CSCs). This flow-cytometry-based protocol allows it to isolate CR-CSCs and to evaluate their cytotoxicity upon anti-tumor treatments. For complete details on the use and execution of this protocol, please refer to Di Franco et al. (2021).

Magnetic Nanoparticle-Based Hyperthermia Mediates Drug Delivery and Impairs the Tumorigenic Capacity of Quiescent Colorectal Cancer Stem Cells.

Cancer stem cells (CSCs) are the tumor cell subpopulation responsible for resistance to chemotherapy, tumor recurrence, and metastasis. An efficient therapy must act on low proliferating quiescent-CSCs (q-CSCs). We here investigate the effect of magnetic hyperthermia (MHT) in combination with local chemotherapy as a dual therapy to inhibit patient-derived colorectal qCR-CSCs. We apply iron oxide nanocubes as MHT heat mediators, coated with a thermoresponsive polymer (TR-Cubes) and loaded with DOXO (TR-DOXO) as a chemotherapeutic agent. The thermoresponsive polymer releases DOXO only at a temperature above 44 °C. In colony-forming assays, the cells exposed to TR-Cubes with MHT reveal that qCR-CSCs struggle to survive the heat damage and, with a due delay, restart the division of dormant cells. The eradication of qCR-CSCs with a complete stop of the colony formation was achieved only with TR-DOXO when exposed to MHT. The in vivo tumor formation study confirms the combined effects of MHT with heat-mediated drug release: only the group of animals that received the CR-CSCs pretreated, in vitro, with TR-DOXO and MHT lacked the formation of tumor even after several months. For DOXO-resistant CR-CSCs cells, the same results were shown, in vitro, when choosing the drug oxaliplatin rather than DOXO and applying MHT. These findings emphasize the potential of our nanoplatforms as an effective patient-personalized cancer treatment against qCR-CSCs.