Cancer

Lecture – Cancer Biology Overview and Disparities:

Overview

- Why is cancer not considered a single disease?

• Cancer is not a single disease as there are over 100 types of cancer each with its own treatment regimen 

• It is defined as a disease resulting from failure of mechanisms that normally control cell growth and proliferation 

- What are the different hallmarks of cancer? (know at least 3; see slides 5 and 6)

- Abnormal morphology 

- Uncontrolled proliferation 

- Loss of contact inhibition

- High telomerase activity

- Chromosomal defects 

- Altered metabolism  

- What are two ways in which cancer cells may LOOK different than normal cells? (e.g, their morphology; see slide 7)

• Cancer cells LOOK different due to their shapes; many cells that grow and divide, and have abnormal number of chromosomes arranged in a disorganized fashion 

- Briefly describe why cancer is considered a “disease of the genome”, with two examples

Cancer is considered a “disease of the genome” due to most tumors harboring a constellation of genomic mutations and alterations EX: 

1. Point mutations, a single nucleotide alteration can result in an amino acid seq of the translated protein product (most common alteration) 

2. Chromosomal abnormalities: gene deletion, amplification or translocation 

→ as a result of these altercations there is a corresponding change in protein activity or expression: 

1. Gain of Function (GoF) mutations/altercations- leads to expression of a mutant protein that is cosistutivly active or increased expression of WT protein (oncogenes) 

2. LOF - leads to expression if protein that is inactive or decreased expression of WT protein (tumor suppressors) 

- Concept that all cancers are genetic, but most are NOT hereditary (slide 19

• Vast majority of cancers are sporadic or familial 

• In hereditary cancer germline mutations exist and will be present in all cells of body 

Disparities

- What are some causes or risk factors for creating cancer health disparities? (be able to list at least 2; slide 25)

1. Genetic and biological factors EX: African american women are nearly twice as likely as white woman to be diagnosed with triple-neg breast cancer and are much more likely than white women to die from breast cancer 

2.  Health Care Access EX: women in rural areas are twice as likely to die from cervical cancer than women in more urban areas 

o What is the difference between health inequality and inequity? slides24)

• Health inequality: difference in health status between populations 

• Health inequity: inequalities that are avoidable, unfair and unjust (Disparities) 

- Why have cancer health disparities been so relatively understudied until recently, and how can they be better addressed? (slides 31-33)

• As of 2018, barely 80% of individuals included in genome research on genetic defects associated with disease risk were of European descent, only 14% of people of color and minorities were accounted for. Meaning that there was a lack of literature and research to account for them. Researchers and medical communities lacked “cultural competence" to effectively interact with people across cultures, whether it be by speaking their language, understanding their backgrounds and providing holistic care. 

• Some other contributions could be lack of transparency with patients, mistrust in medical research, perceived discrimination, communication barriers 

• We can address cancer health care disparities by improving access to preventive and screening measures, include samples and genomic data from diverse populations, include patient advocates in scientific studies, engage and recruit minority participants in trials- major focus of current research 

Lecture – Tumor Microenvironment + Immunology

Microenvironment

- What are the different types of cells commonly found in the tumor

microenvironment (both cancerous and non-cancerous)?; be able to briefly

describe 2 cancer cell examples and 2 non-cancerous cell examples (slide 5)

• The microenvironment can affect how a tumor grows and spreads 

• The tumor microenvironment (TME) is a complex ecosystem of cells, molecules, and blood vessels that surround and support a tumor.

• Cancerous cells: 

  • Bulk Cancer cells: most common type of cancer cell in TME, responsible for bulk of tumors growth 

  • Cancer stem cells: ability to self-renewal and differentiate into other types of cancer cells 

• Non Cancerous: 

  • Lymphocytes: specifically T lymphocytes -> kill cancer cells directly 

  • Macrophages: can be either pro or anti tumor depending on the signals given by TME

o Drawing it out helps! (slide 4)

- What are some ways in which cancer cells cooperate in the tumor

microenvironment to support tumorigenesis

• Cancer cells can interact with fibroblasts in TME, as they secrete a variety of factors into TME that can affect tumor behavior and promote tumor growth (tumorigenesis), recruiting  more fibroblasts telling to make new blood vessels 

• Making more of an extravell matrix from drugs tyring to get in 

→  EX: can release growth factors and matrix metalloproteinases that contribute  which contribute to tumor proliferation and invasion, also tumor neoangiogenesis, immunosuppression

• Adipose tissue: Cancer cells can utilize resources in adipose tissues TME to support growth and metabolism; Adipose tissue releases cytokines and fatty acids, which can be used by cancer cells for energy production and building blocks for cell division → pro inflam mediators, cell proliferation and growth 

• EMT: Cancer cells can secrete chemokines and other soluble factors that induce EMT in neighboring cells and enable site specific metastasis 

  • EMT is a process where epithelial cells lose their cell-cell adhesion and gain migratory and invasive properties, contributing to metastasis

o What are some of the broad roles of cytokines and/or chemokines within the TME or for promoting cancer growth?

• Cytokines play a crucial role in regulating the activity of immune cells in TME, they can either stimulate or suppress immune responses 

  • EX: TNFa and IL-2

• Promotion of Tumor growth and Angiogenesis: certain cytokines and chemokines act as growth factors directly stimulating the proliferation of cancer cells, promote angiogenesis, formation of new blood vessels 

  • EX: VEGF is a potent angiogenic factor 

• Induction of EMT and Metastasis: chemokines and other soluble factors can induce EMT in cancer cells 

• Shaping and TME composition; cytokines and chemokines can influence recruitment and differentiation of various cell types into TME

• Secreted soluble factors can secrete chemokines in the TME, secrete growth factors, angiotensin

• Slide 11, cancer cells that start to escape will express c ertian receptors to get on endothelial cells into blood vessels and travel, some tissues have natural high levels 

• Fibroblasts secrete a variety of factors into TME, both host cells can secret cytokines, promote more tumorigenesis metastasis escaping, suppressing immune response  

- How could you identify the types of subtypes of cancer cells present in a tumor? (slides 7; 14): be able to discuss one type of experimental approach

• RNA -seq or scRNA-Seq: 

  • The tumor sample is first dissociated into individual cells for analysis of gene expression at single cell level 

  • RNA is then extracted from the isolated cells and sequences, RNA seq determines the seq of RNA molecules making a snapshot of actively transcribed genes

  • The then seq data is then analyzed forming gene clusters that represent diff subtypes of cancer cells w/n tumor , then they can identify molecular markers that distinguish diff cancer cell subtypes  

o Types of cytokines or genes/proteins expressed? (slides 12-23)

• Certain cytokines are indicative of certain types of tumors, identify cells and what they make, cytokine arrays to qualitative secreted factors, have antibodies that are specific to cytokine or growth of interest, antibodies, cell lysate sample, run through, and whatever binds to antibody of choice, use a second set if antibody conjugated to fluorophore or enzyme, understand what cytokines are in sample; show diff shapes of cancerous cells 

• Morphology of cancer and detect immunohistochemical staining 

• Flow cytometry where bulk tumor cell, cell suspension removal of dead cells, to tell flow cytometry; on outside, profile diff cell types by what they express 

• RNA-SEQ, at single cell level, who is expressing what in tumor biopsy EX: sub types cancer slide 

Question on exam, 

Immunology

- What are 2 ways the immune system can control or inhibit cancer (broadly speaking; see slide 16)

• Destroys viruses that are known to transform cells , so no cancer cells 

  • Aka oncogene that help proliferate non stop  

• Eliminates pathogens to regulate inflammation 

  • Chronic cytokine sig, damage to tissue, high cortisol, high stress → helps suppress tumors 

- What is the main difference between DAMPs and PAMPs in their ability to activate the immune system? (slide 19)

• DAMPs: are HOST FACTORS, like heat shock proteins, ECM proteins, DNA, RNA and metabolites are released by dying cancer cells due to necrosis  meanwhile PAMPS are molecular structures found on pathogens like bacteria and viruses that are recognized by PRRS on cells 

- Be able to list 1 way a macrophage can kill a cancer cell (slides 22-24)

• A macrophage can kill a cancer cell by direct killing through release of harmful products such as nitric oxide or TNF

  • ROS can directly kill tumor, macrophages can do this as they recognize DAMP

- What is 1 way natural killer (NK) cells are naturally activated to kill tumor cells?

• Over Regulation of stress induced ligands promotes killing of tumor cells aka stress induced self as it creates a potent trigger for NK cell activation since they have none or too much inhibitory signals causing an imbalance 

- How can we use immunotherapy to enhance the cytotoxicity of NK cells? Of T cells?

o Be able to draw/describe one of these approaches in a short answer (see slides 28-20)

• For NK cells we can use Adoptive cell transfer where in a cancer patient PBMCs from blood are taken for ex-vivo expansion ( goes from 2x10^5 to 1x10^9 takes ab 2-3 weeks) (selecting certain type) which are then primed and reprogrammed  to highly activated NK cells, which are then transferred back into the patient to increase the number of NK cells targeting tumor cells 

• In T cells it is similar, involving enriching for tumor antigen specific dendritic cells or T cells, plate fragments, asses T cells, expand T cells and get reinfused to the patient to enhance anti-tumor cells 

DO NOT STUDY: 

• CAR T cell therapy: create modified T cell receptor that will recognize receptors from your tumor 

• Design a receptor that will recognize antigen in pts tumor, function like a T-cell receptor, reinfuse them and go back to pts 

- Describe one way a tumor may start to escape targeting by the immune system

• By expressing PD-L1 on its surface, PD-L1 is a ligand for PD-1 receptor which is a neg reg of T-cells, When PD-L1 on a tumor cell binds to PD-1 on a T cell, it inhibits the T cell's cytotoxic activity and interferon-γ production, effectively suppressing the immune response against the tumor

• Tumor cells engineered to express PD-L1 were less susceptible to killing by cytotoxic T cells compared to parental cells lacking PD-L1, leading to the tumor escaping and inducing enhanced tumor growth and proliferation 

• EX: In mice, tumor cells expressing PD-L1 showed enhanced tumor growth and invasiveness compared to control cells. This effect was dependent on the presence of T cells, indicating that PD-L1 expression allowed the tumor to escape immune surveillance

• Can evolve ways to hide, secrete specific cytokines to evade the , more general, most likely multiple choice question 

- Why are antibodies against CTLA-4 or PD-1 good for cancer treatment?

o Slides 38-39; Paper 3

• Activated by TCR sig, if they continue to be activates, it triggers expression and localization of inhibitory receptors 

• Once CTLA-4 is activated it outcompetes and binds, leading to repression of T cell activity 

• PD1-L, instead of T cell itsel;f doing autoreg, cancer cell tries to figure out the pumping the brake signals 

• Antibodies against CTLA-4 or PD-1 are effective for cancer treatment because they block the negative regulation of T cells, allowing for a stronger anti-tumor immune response

• CTLA-4 binds to B7 ligands while PD-1 interacts with PD-1 L1 and PD-L2

• When CTLA-4 or PD-1 are engaged by their ligands, the T cell response is inhibited; Tumor cells can exploit these negative immunoreceptors to evade the immune system

• CTLA-4 and PL-1 are natural checkpoints, tumor cells can upregulate ligands that bind to these suppressing activity ot tumor fighting T-cells, the antibodies against CTLA-4 or PD-1 block the binding of CTLA-4 or PD-1 to their ligands releasing their brakes on the immune system allowing T cells to become more active and target tumor cells  

• CTLA-4 blockade acts like pulling up the parking brake while PD-1 blockade acts as pumping the brakes 

Paper 3

• CTLA-4 blockade enhances tumor immunity by preventing the restriction of T cell activation and expansion, primarily through cross-presentation of tumor antigens by host antigen-presenting cells.

• PD-L1 expression on tumor cells directly protects them from attack by cytotoxic T cells.

  • This was demonstrated by reduced cytotoxicity and interferon-γ production by T cells against tumor cells expressing PD-L1.

  • Blocking PD-L1 with antibodies restored T cell function and inhibited tumor growth in mice.

• PD-L1 expression on tumor cells also enhances tumorigenicity and invasiveness.

  • This is likely due to the inhibition of both the effector phase of the T cell response and potentially T cell priming.

Therefore, blocking CTLA-4 or PD-1 with antibodies removes the negative regulation of T cells, unleashing a stronger anti-tumor immune response that can lead to tumor suppression and rejection

o How would you test a drug against cancer in a mouse model In-vitro?

1. Isolate immune cells and tumor cells from mice; ex CTLS from spleens of mice, culture mouse tumors that express PD-L1

2. Establish Co-culture ; set up co-cultures of the isolated mouse CTLS and tumor cells in vitro, allowing for direct interaction between immune cells and tumor cells mimicking the TME 

3. Treat co-cultures with drug candidate: introduce anti-cancer drug candidate aka an antibody 

4. Asses the Drug Effects on tumor cell viability and immune cell function 

5. Include control groups