In Vivo Tests for Anticancer Activity

Why In Vivo Testing Is Needed

• Drug absorption, distribution, metabolism, and excretion (ADME)
• Tumor microenvironment interactions (vasculature, immune cells, stroma)
• Toxicity at therapeutic doses
• Whether effective drug concentrations are achievable in intact tissue
• Tumor growth delay, regression, and cure rates

Categories of In Vivo Models

1. Carcinogen-Induced Models

• DMBA (7,12-dimethylbenz[a]anthracene) - induces mammary tumors in rats
• Azoxymethane (AOM) - induces colon cancer in rodents
• Benzo[a]pyrene - induces lung tumors
• N-nitrosodiethylamine (NDEA) - hepatocellular carcinoma model

• Tumors develop naturally, mimicking the multi-step carcinogenic process
• Immunocompetent host - immune response is intact
• Useful for chemoprevention studies

• Long latency period (weeks to months)
• Variable tumor incidence and time of onset
• Not suitable for rapid drug screening

2. Transplantation Models

a) Syngeneic (Allograft) Models

• Rodent tumor cells are transplanted into genetically identical (inbred strain) immunocompetent mice
• Examples: LLC (Lewis Lung Carcinoma) in C57BL/6 mice; B16 melanoma; 4T1 breast cancer in BALB/c mice
• Advantage: Intact immune system - good for immunotherapy studies
• Limitation: Rodent tumors may not reflect human tumor biology

b) Xenograft Models

• Human tumor cells are implanted into immunodeficient mice (nude mice, SCID mice, NSG mice) to prevent rejection

• Use actual human tumor cells
• Subcutaneous tumors easily measurable
• Reproducible

• Immunodeficient host lacks a normal immune response
• Species difference in drug sensitivity can cause artifacts (e.g., cardiac glycosides kill human cells but not rodent cells, giving false-positive anticancer results in xenograft models)
• Poor predictor of clinical outcomes if the host biology is very different

3. Viral Infection / Viral-Induced Models

• Moloney murine leukemia virus - induces lymphoma/leukemia
• Rous sarcoma virus - fibrosarcoma in chickens
• Hepatitis B virus (woodchuck model) - hepatocellular carcinoma

4. Genetically Engineered Mouse Models (GEMMs)

• MMTV-neu mice - HER2-overexpressing mammary tumors
• APC^Min/+ mice - develop intestinal polyps (colon cancer model)
• KRAS^G12D mice - lung and pancreatic cancer models
• p53 knockout mice - develop sarcomas and lymphomas

• Tumors arise in an immunocompetent, genetically defined host
• Recapitulate the genetic alterations seen in human cancers
• Ideal for targeted therapy testing

• Expensive and time-consuming to generate
• May not perfectly mirror human tumor heterogeneity

5. In Vivo Hollow Fiber Assay (HFA)

• 12 human tumor cell lines (lung, breast, colon, melanoma, ovary, glioma) are suspended inside hollow polyvinylidene fluoride (PVDF) fibers
• Fibers are implanted intraperitoneally (IP) or subcutaneously (SC) in immunodeficient mice
• The drug is administered to the live mouse
• After treatment (typically a few days), fibers are removed and analyzed in vitro for cell viability / growth inhibition

• Tests multiple tumor types simultaneously in one animal
• Bridges in vitro and in vivo - drug must reach the fiber in vivo
• Faster and uses fewer animals than traditional xenograft models
• Good for prioritizing which compounds merit full xenograft testing

• Tumor cells are not fully integrated into host tissue
• No tumor vascularity or stroma interactions

Key Endpoints Measured in In Vivo Anticancer Tests

Tumor Growth Delay Assay

• Tumor regrowth is monitored
• Growth delay = difference in time to reach a defined volume between treated and control animals
• Enables calculation of log cell kill and assessment of curative potential
• Validates drug activity for clinical trial design

Important Limitations of In Vivo Models

1. Species differences: Rodent cells can be highly resistant or sensitive to certain drugs compared to human cells, giving misleading results (classic example: cardiac glycosides appear anticancer in xenografts but fail clinically)
2. Tumor microenvironment: Subcutaneous ectopic tumors lack the native vasculature, stroma, and immune infiltrates of the original tumor site
3. Immune status: Xenograft models require immunodeficient mice, masking immunotherapy effects
4. Metastasis: Most standard models use primary tumor endpoints; metastatic models are more predictive for most clinical scenarios since the majority of cancer patients have metastatic disease
5. High cost and animal welfare concerns

Summary: Hierarchy of In Vivo Tests

Carcinogen-induced models     → Chemoprevention & natural carcinogenesis
Viral-induced models          → Virus-associated cancers
GEMMs                         → Targeted therapy, oncogene-driven cancers
Syngeneic transplant models   → Immunotherapy, intact immune system
In Vivo Hollow Fiber Assay    → Rapid multi-tumor screening
Xenograft models (ectopic)    → Standard efficacy screening (human cells)
Orthotopic xenografts         → Clinically relevant site-specific testing
Metastasis models             → Most translatable to clinical disease

In Vivo Tests for Anticancer Activity of Plant Extracts

General Protocol Framework

Plant material → Extraction (Soxhlet / maceration)
       ↓
Phytochemical screening (alkaloids, flavonoids, terpenes, etc.)
       ↓
In vitro cytotoxicity (MTT, SRB, trypan blue on cancer cell lines)
       ↓
In vivo animal models (below)
       ↓
Mechanism studies (apoptosis, angiogenesis, oxidative stress)
       ↓
Toxicity & safety profiling

Common In Vivo Models Used for Plant Extracts

1. Ehrlich Ascites Carcinoma (EAC) Model

• Ehrlich tumor cells (originally a mammary adenocarcinoma) are maintained as an ascitic (fluid) tumor in Swiss albino mice by serial intraperitoneal (IP) passage
• A set volume of EAC cells (e.g., 1 × 10⁶ cells/mouse) is injected IP into mice
• Plant extract is administered (usually orally or IP) daily for 14 days starting 24 hours after tumor inoculation
• Standard positive control: 5-Fluorouracil (5-FU) or Cyclophosphamide

• % ILS ≥ 25% = significant anticancer activity (NCI criterion)
• A good plant extract typically shows % ILS of 30-80% in well-designed studies

• Fast (results in ~14 days)
• Cheap, reproducible
• Well-standardized protocol
• Good for screening crude extracts

2. Dalton's Lymphoma Ascites (DLA) Model

3. Sarcoma 180 (S-180) Solid Tumor Model

• S-180 cells injected SC into the flank
• Extract administered for 10-14 days
• Tumor dissected and weighed on the final day

• Tumor weight (g) - primary endpoint
• Tumor growth inhibition (%) = [(Wcontrol - Wtreated) / Wcontrol] × 100
• Tumor volume (caliper measurement: V = L × W² / 2)
• Organ weights (spleen, liver, thymus) for immune/toxicity assessment

4. Hepatoma 22 (H22) / Lewis Lung Carcinoma (LLC) / B16 Melanoma Models

• H22 (hepatoma) - liver cancer model in immunocompetent mice; particularly used for plants with hepatoprotective-anticancer dual activity
• LLC (Lewis Lung) - C57BL/6 mice; used when plant extract is targeted at lung cancer
• B16 melanoma - C57BL/6 mice; for skin/melanoma-targeting plant extracts
• 4T1 breast cancer - syngeneic BALB/c model for breast cancer-active plants

5. Carcinogen-Induced Models

6. Xenograft Models (Human Tumor - Athymic Nude Mice)

• Human cancer cells (e.g., MCF-7, HCT-116, A549, PC-3) injected SC into nu/nu (nude) mice or SCID mice
• Plant extract or isolated phytochemical administered IP or orally once tumor reaches ~100-200 mm³
• Treatment continued 3-4 weeks

• Tumor volume (caliper, twice weekly)
• Tumor growth inhibition (TGI %)
• Relative tumor volume (RTV)
• T/C ratio (treated / control tumor volume)
• Mouse body weight (toxicity)
• Ki-67 (proliferation marker), TUNEL (apoptosis) on excised tumor sections

7. In Vivo Hollow Fiber Assay (HFA)

• 12 human tumor cell lines in PVDF hollow fibers implanted IP/SC in mice
• Particularly useful for plant extracts where the active component is unknown and multiple tumor types need screening
• Plant extract administered; fibers retrieved after a few days and analyzed in vitro

Parameters Routinely Measured Across All In Vivo Models

Tumor-Related

• Tumor volume / weight / ascitic fluid volume
• Viable and non-viable tumor cell counts (trypan blue)
• % Tumor Growth Inhibition (TGI)
• % Increase in Life Span (% ILS) - ascitic models
• Mean Survival Time (MST)

Hematological (very commonly reported for plant extract studies)

• RBC, Hemoglobin, Hematocrit - EAC/DLA causes anemia; reversal indicates activity
• WBC - tumor causes leukocytosis; normalization is a positive sign
• Platelet count
• Differential count (lymphocytes, neutrophils)

Biochemical / Hepatic

• SGPT (ALT), SGOT (AST) - liver function
• ALP, Total bilirubin
• Serum proteins, Albumin
• Triglycerides, Cholesterol - tumor-induced dyslipidemia

Oxidative Stress Markers (unique feature of plant extract studies)

• MDA (Malondialdehyde) - lipid peroxidation marker; elevated in tumor-bearing mice; reduced by effective extract
• GSH (Glutathione) - antioxidant; depleted in tumor mice; restored by extract
• SOD (Superoxide Dismutase) - antioxidant enzyme
• Catalase - antioxidant enzyme

Histopathology

• Liver, kidney, spleen sections (H&E stain) for toxicity assessment
• Tumor tissue sections for necrosis, apoptotic bodies
• Immunohistochemistry: Ki-67 (proliferation), Bcl-2/Bax (apoptosis), VEGF (angiogenesis), Caspase-3

Immunological Parameters (increasingly assessed)

• IFN-γ (elevated = immune activation)
• IL-4, IL-6, TNF-α levels
• Spleen/thymus weight and lymphocyte counts
• NK cell activity - particularly for polysaccharide-rich plant extracts

Typical Experimental Groups for EAC/DLA Studies

Calculation of Key Efficacy Indices

% ILS = [(MST treated - MST control) / MST control] × 100

TGI% = [(Wcontrol - Wtreated) / Wcontrol] × 100

CGI% = [(viable cells control - viable cells treated) / viable cells control] × 100

Special Considerations for Plant Extracts

1. Crude vs. fractionated extract: In vivo studies are often done with crude extracts first, then repeated with bioactive fractions to identify the active phytochemical(s)
2. Route of administration: Oral (gavage) is preferred as it reflects traditional use; IP injection is used for rapid screening
3. Dose selection: Based on prior acute toxicity studies (LD₅₀ from OECD 423 or 425 guidelines); typically 1/10th to 1/5th of LD₅₀ is used
4. Vehicle: Extracts are often dissolved in DMSO (1%), CMC (0.5%), or distilled water depending on solubility
5. Phytochemical synergy: Unlike single pure compounds, plant extract activity may result from synergy between flavonoids, alkaloids, terpenoids, and polyphenols - making dose-response curves non-linear
6. Immunostimulation vs. direct cytotoxicity: Some plant polysaccharides and glycosides work primarily by boosting NK cells and macrophage activity rather than directly killing tumor cells - immune parameters must be assessed

Summary Flowchart

Plant extract (crude)
        ↓
Acute toxicity study (LD₅₀) → select safe doses
        ↓
EAC / DLA ascitic model → % ILS, tumor volume, hematology
        ↓
S-180 solid tumor model → TGI%, tumor weight
        ↓
Carcinogen model → if chemoprevention claim
        ↓
Xenograft (nude mice) → if specific human cancer cells targeted
        ↓
Mechanism studies → apoptosis, oxidative stress, angiogenesis, immunology
        ↓
Fractionation → isolate active phytochemical
        ↓
Repeat in vivo with pure compound