Cancer Biology

Over the past decades, the main techniques used to visualize bacteria in tissue have improved but are still mainly based on indirect recognition of bacteria. Both microscopy and molecular recognition are being improved, but most procedures for bacteria detection in tissue involve extensive damage. Here, we describe a method to visualize bacteria in tissue slices from an in vivo model of breast cancer. This method allows examining trafficking and colonization of fluorescein-5-isothiocyanate (FITC)-stained bacteria in various tissues. The protocol provides direct visualization of fusobacterial colonization in breast cancer tissue. Rather than processing the tissue or confirming bacterial colonization by PCR or culture, the tissue is directly imaged using multiphoton microscopy. This direct visualization protocol causes no damage to the tissue; therefore, all structures can be identified. This method can be combined with others to co-visualize bacteria, types of cells, or protein expression in cells.

Bone metastasis is a frequent and lethal complication of many cancer types (i.e., prostate cancer, breast cancer, and multiple myeloma), and a cure for bone metastasis remains elusive. To recapitulate the process of bone metastasis and understand how cancer cells metastasize to bone, intracardiac injection and intracaudal arterial animal models were developed. The intratibial injection animal model was established to investigate the communication between cancer cells and the bone microenvironment and to mimic the setting of prostate cancer patients with bone metastasis. Given that detailed protocols of intratibial injection and its quantitative analysis are still insufficient, in this protocol, we provide hands-on procedures for how to prepare cells, perform the tibial injection, monitor tibial tumor growth, and quantitatively evaluate the tibial tumors in pathological samples. This manuscript provides a ready-to-use experimental protocol for investigating cancer cell behaviors in bone and developing novel therapeutic strategies for bone metastatic cancer patients.

Characterization of key regulators in vein development will advance our understanding of mechanisms underlying venous anomalies and provide therapeutic targets for the treatment of vascular malformations. Here, we provide a detailed protocol for the generation of genetically engineered mouse models targeting the Tek gene for the analysis of vein formation and vein-associated vascular diseases at the embryonic and postnatal stages. It includes steps involved in the whole-mount processing of mouse skin, mesentery, and retina for the examination of vascular malformation during embryonic and postnatal development.

The mammary gland is a highly dynamic tissue that changes throughout reproductive life, including growth during puberty and repetitive cycles of pregnancy and involution. Mammary gland tumors represent the most common cancer diagnosed in women worldwide. Studying the regulatory mechanisms of mammary gland development is essential for understanding how dysregulation can lead to breast cancer initiation and progression. Three-dimensional (3D) mammary organoids offer many exciting possibilities for the study of tissue development and breast cancer. In the present protocol derived from Sumbal et al., we describe a straightforward 3D organoid system for the study of lactation and involution ex vivo. We use primary and passaged mouse mammary organoids stimulated with fibroblast growth factor 2 (FGF2) and prolactin to model the three cycles of mouse mammary gland lactation and involution processes. This 3D organoid model represents a valuable tool to study late postnatal mammary gland development and breast cancer, in particular postpartum-associated breast cancer.

Graphic abstract:

Mammary gland organoid isolation and culture procedures

Tumor xenograft models developed by transplanting human tissues or cells into immune-deficient mice are widely used to study human cancer response to drug candidates. However, immune-deficient mice are unfit for investigating the effect of immunotherapeutic agents on the host immune response to cancer (Morgan, 2012). Here, we describe the preparation of an orthotopic, syngeneic model of lung adenocarcinoma (LUAD), a subtype of non-small cell lung cancer (NSCLC), to study the antitumor effect of chemo and immunotherapeutic agents in an immune-competent animal. The tumor model is developed by implanting 344SQ LUAD cells derived from the metastases of Kras^G12D; p53^R172HΔG genetically engineered mouse model into the left lung of a syngeneic host (Sv/129). The 344SQ LUAD model offers several advantages over other models: 1) The immune-competent host allows for the assessment of the biologic effects of immune-modulating agents; 2) The pathophysiological features of the human disease are preserved due to the orthotopic approach; 3) Predisposition of the tumor to metastasize facilitates the study of therapeutic effects on primary tumor as well as the metastases (Chen et al., 2014). Furthermore, we also describe a treatment strategy based on Poly(2-oxazoline) micelles that has been shown to be effective in this difficult-to-treat tumor model (Vinod et al., 2020b).

Metastasis accounts for the majority of cancer related deaths. The genetically engineered mouse (GEM) models and cell line-based subcutaneous and orthotopic mouse xenografts have been developed to study the metastatic process. By using lung cancer cell line A549 as an example, we present a modified protocol to establish the cell line-based xenograft. Our protocol ensures sufficient establishment of the mouse xenografts and allows us to monitor tumor growth and spontaneous metastasis. This protocol could be adapted to other types of established cancer cell lines or primary cancer cells to study the mechanism of metastatic process as well as to test the effect of the potential anti-cancer agents on tumor growth and metastatic capacity.

Various genetic alterations such as chromosomal translocation cause leukemia. For examples, gene rearrangements of the mixed-lineage leukemia (MLL) gene generate MLL fusion genes, whose products are potent oncogenic drivers in acute leukemia. To better understand the mechanism of disease onset, several murine leukemia models using retroviral gene transduction, xenograft, or Cre-mediated chromosomal translocation have been developed over the past twenty years. Particularly, a retroviral gene transduction-mediated murine leukemia model has been frequently used in the leukemia research field. Here, we describe the detailed protocol for this model.

Uveal melanoma (UM) is a malignant intraocular tumor in adults. Metastasis develops in almost half of the patients and over 90% of the metastases are in the liver. With the advances in molecular targeting therapy for melanoma, a proper metastasis animal model is of increasing importance for testing the accuracy and effectiveness of systemic therapies. Here, we describe a xenograft model for mimicking human UM liver metastasis by injecting human UM cells into the vitreous cavity in nude mice. The athymic nude mice are immunocompromised and suitable for xenograft tumor growth and metastasis, and intravitreal injection of cells is a quicker and easier operation under a binocular scope, thereby it is simple and effective to test human UM growth and metastasis.

Glioblastoma (GBM) is the most common primary brain cancer in adults and has a poor prognosis. It is characterized by a high degree of cellular infiltration that leads to tumor recurrence, atypical hyperplasia, necrosis, and angiogenesis. Despite aggressive treatment modalities, current therapies are ineffective for GBM. Mouse GBM models not only provide a better understanding in the mechanisms of gliomagenesis, but also facilitate the drug discovery for treating this deadly cancer. A retroviral vector system that expresses PDGFBB (Platelet-derived growth factor BB) and inactivates PTEN (Phosphatase and tensin homolog) and P53 tumor suppressors provides a rapid and efficient induction of glioma in mice with full penetrance. In this protocol, we describe a simple and practical method for inducing GBM formation by retrovirus injection in the murine brain. This system gives a spatial and temporal control over the induction of glioma and allows the assessment of therapeutic effects with a bioluminescent reporter.

To investigate whether endothelial Akt1 activation is sufficient to induce vascular tumor formation in the skin, we have developed a skin graft model in which a skin fragment from transgenic donor mice with inducible and endothelial cell-specific overexpression of activated Akt1 (myrAkt1) is grafted into the skin of wild type recipient mice. The donor skin successfully engrafts after two weeks and, more importantly, vascular tumor develops at the site of transgenic skin graft when myrAkt1 expression is turned on. This skin graft model is a novel approach to investigate the biological impact of a key signal transduction molecule in a temporal, localized and organ-specific manner.