Pancreatic ductal adenocarcinoma (PDA) was responsible for ~ 44,000 deaths in the United States in 2018 and is the epitome of a recalcitrant cancer driven by a pharmacologically intractable ...oncoprotein, KRAS
. Downstream of KRAS, the RAF→MEK→ERK signaling pathway plays a central role in pancreatic carcinogenesis
. However, paradoxically, inhibition of this pathway has provided no clinical benefit to patients with PDA
. Here we show that inhibition of KRAS→RAF→MEK→ERK signaling elicits autophagy, a process of cellular recycling that protects PDA cells from the cytotoxic effects of KRAS pathway inhibition. Mechanistically, inhibition of MEK1/2 leads to activation of the LKB1→AMPK→ULK1 signaling axis, a key regulator of autophagy. Furthermore, combined inhibition of MEK1/2 plus autophagy displays synergistic anti-proliferative effects against PDA cell lines in vitro and promotes regression of xenografted patient-derived PDA tumors in mice. The observed effect of combination trametinib plus chloroquine was not restricted to PDA as other tumors, including patient-derived xenografts (PDX) of NRAS-mutated melanoma and BRAF-mutated colorectal cancer displayed similar responses. Finally, treatment of a patient with PDA with the combination of trametinib plus hydroxychloroquine resulted in a partial, but nonetheless striking disease response. These data suggest that this combination therapy may represent a novel strategy to target RAS-driven cancers.
Development and preclinical testing of new cancer therapies is limited by the scarcity of in vivo models that authentically reproduce tumor growth and metastatic progression. We report new models for ...breast tumor growth and metastasis in the form of transplantable tumors derived directly from individuals undergoing treatment for breast cancer. These tumor grafts illustrate the diversity of human breast cancer and maintain essential features of the original tumors, including metastasis to specific sites. Co-engraftment of primary human mesenchymal stem cells maintains phenotypic stability of the grafts and increases tumor growth by promoting angiogenesis. We also report that tumor engraftment is a prognostic indicator of disease outcome for women with newly diagnosed breast cancer; orthotopic breast tumor grafting is a step toward individualized models for tumor growth, metastasis and prognosis. This bank of tumor grafts also serves as a publicly available resource for new models in which to study the biology of breast cancer.
Research models that replicate the diverse genetic and molecular landscape of breast cancer are critical for developing the next-generation therapeutic entities that can target specific cancer ...subtypes. Patient-derived tumorgrafts, generated by transplanting primary human tumor samples into immune-compromised mice, are a valuable method to model the clinical diversity of breast cancer in mice, and are a potential resource in personalized medicine. Primary tumorgrafts also enable in vivo testing of therapeutics and make possible the use of patient cancer tissue for in vitro screens. Described in this unit are a variety of protocols including tissue collection, biospecimen tracking, tissue processing, transplantation, and three-dimensional culturing of xenografted tissue, which enable use of bona fide uncultured human tissue in designing and validating cancer therapies.
A unique alkene difunctionalization reaction that allows rapid construction of molecular complexity around the biologically relevant indole framework has been developed. The reaction proceeds with up ...to 87% yield, 99:1 er, and >20:1 dr. Evaluation of several of the compounds revealed promising anticancer activity against MCF-7 cells.
The mouse mammary gland is the only epithelial organ capable of complete regeneration upon orthotopic transplantation, making it ideally suited for in vivo gene function studies through ...viral-mediated gene delivery. A hurdle that has challenged the widespread adoption of this technique has been the inability to transduce mammary stem cells effectively. We have overcome this limitation by infecting total primary mammary epithelial cells in suspension with high-titer lentiviruses. Transduced cells gave rise to all major cell types of the mammary gland and were capable of clonal outgrowth and functional differentiation in serial transplants. To demonstrate that this method is a valuable alternative to developing transgenic animals, we used lentiviral-mediated Wnt-1 overexpression to replicate MMTV-Wnt-1 mammary phenotypes and used a dominant-negative Xenopus Suppressor of Hairless to reveal a requirement for Notch signaling during ductal morphogenesis. Importantly, this method is also applicable to transduction of cells from other tissues.
Toward improved models of human cancer Welm, Bryan E.; Vaklavas, Christos; Welm, Alana L.
APL bioengineering,
03/2021, Volume:
5, Issue:
1
Journal Article
Peer reviewed
Open access
Human cancer is a complex and heterogeneous collection of diseases that kills
more than 18 million people every year worldwide. Despite advances in detection,
diagnosis, and treatments for cancers, ...new strategies are needed to combat
deadly cancers. Models of human cancer continue to evolve for preclinical
research and have culminated in patient-derived systems that better represent
the diversity and complexity of cancer. Still, no model is perfect. This
Perspective attempts to address ways that we can improve the clinical
translatability of models used for cancer research, from the point of view of
researchers who mainly conduct cancer studies in vivo.
Defining master transcription factors governing somatic and cancer stem cell identity is an important goal. Here we show that the Oct4 paralog Oct1, a transcription factor implicated in stress ...responses, metabolic control, and poised transcription states, regulates normal and pathologic stem cell function. Oct1(HI) cells in the colon and small intestine co-express known stem cell markers. In primary malignant tissue, high Oct1 protein but not mRNA levels strongly correlate with the frequency of CD24(LO)CD44(HI) cancer-initiating cells. Reducing Oct1 expression via RNAi reduces the proportion of ALDH(HI) and dye efflux(HI) cells, and increasing Oct1 increases the proportion of ALDH(HI) cells. Normal ALDH(HI) cells harbor elevated Oct1 protein but not mRNA levels. Functionally, we show that Oct1 promotes tumor engraftment frequency and promotes hematopoietic stem cell engraftment potential in competitive and serial transplants. In addition to previously described Oct1 transcriptional targets, we identify four Oct1 targets associated with the stem cell phenotype. Cumulatively, the data indicate that Oct1 regulates normal and cancer stem cell function.
3D patient tumor avatars (3D-PTAs) hold promise for next-generation precision medicine. Here, we describe the benefits and challenges of 3D-PTA technologies and necessary future steps to realize ...their potential for clinical decision making. 3D-PTAs require standardization criteria and prospective trials to establish clinical benefits. Innovative trial designs that combine omics and 3D-PTA readouts may lead to more accurate clinical predictors, and an integrated platform that combines diagnostic and therapeutic development will accelerate new treatments for patients with refractory disease.
3D patient tumor avatars (3D-PTAs) hold promise for next-generation precision medicine. Here, we describe the benefits and challenges of 3D-PTA technologies and necessary future steps to realize their potential for clinical decision making. 3D-PTAs require standardization criteria and prospective trials to establish clinical benefits. Innovative trial designs that combine omics and 3D-PTA readouts may lead to more accurate clinical predictors, and an integrated platform that combines diagnostic and therapeutic development will accelerate new treatments for patients with refractory disease.
Patient-derived xenografts (PDXs) have clinical value but are time-, cost-, and labor-intensive and thus ill-suited for large-scale experiments. Here, we present a protocol to convert PDX tumors into ...PDxOs for long-term cultures amenable to moderate-throughput drug screens, including in-depth PDxO validation. We describe steps for PDxO preparation and mouse cell removal. We then detail PDxO validation and characterization and drug response assay. Our PDxO drug screening platform can predict therapy response in vivo and inform functional precision oncology for patients.
For complete details on the use and execution of this protocol, please refer to Guillen et al.1
Display omitted
•Protocol to establish long-term breast cancer organoids from patient-derived xenografts•Characterization and validation of PDxOs•Detailed steps for efficient PDxO drug screening•Adaptable for co-clinical functional precision oncology studies
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Patient-derived xenografts (PDXs) have clinical value but are time-, cost-, and labor-intensive and thus ill-suited for large-scale experiments. Here, we present a protocol to convert PDX tumors into PDxOs for long-term cultures amenable to moderate-throughput drug screens, including in-depth PDxO validation. We describe steps for PDxO preparation and mouse cell removal. We then detail PDxO validation and characterization and drug response assay. Our PDxO drug screening platform can predict therapy response in vivo and inform functional precision oncology for patients.
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