IMMUNOTHERAPY AND IMMUNE MONITORING
INTRODUCTION, SINGLE CELL MODALITIES 5-9-2023
Goal of the course: becoming familiar quantification
and monitoring of immune responses
Paper discussed during WG and presentations are not
part of the exam, open-ended exam questions
If you want to reach course coordinator use
amsterdamumc email
Immunotherapy: a type of treatment in which the
immune system is modulated (enhanced or
suppressed)
with the ultimate goal of curing/alleviating a disease
The immune system has tremendous heterogeneity, how do different cell types contribute in different tissues
→ needs to be studied on a single cell level to completely understand
Biological systems are composed of heterogeneous cell types in which minority cell populations play important
roles:
- in many tumours, only a small infraction (typically <1%) of molecularly distinct cells called ‘Cancer Stem Cells’
have the capacity for self-renewal and tumorigenesis → understanding these cells has an enormous
therapeutic potential
- a minority of tissue-specific stem cell populations are responsible for tissue development, regeneration and
repair
- the immune system is a classic example for the need of single cell technologies, because its dynamic cellular
heterogeneity is essential for fighting off the variety of attacking pathogens
Bulk techniques:
- RNA sequencing
- PCR
- Western blotting
- proteomics
Single cell techniques:
- flow cytometry
- single cell RNA sequencing
- microscopy – observing single cells using segmentation, masks
Understanding tissue heterogeneity:
mTOR targets phospho-ribosomal protein S6 and phosphor-4E-BP1 exhibit substantial
cellular exclusivity in colorectal cancer speciments, with rare co-expression
- rows (A-C) show different patterns of mutually exclusive p4E-BP1 (red) and pS6
(green) signals
- (A1-A3) many specimens exhibit a strong signal for each phosphorylation event in
exclusive tumour cells
- (A4-C2) another notable pattern exhibits substantial p4E-BP1 expression in tumour
cells, and a high level pS6 signal in stromal cells
- (C3-C5) rare tumours exhibit high level signal in only one of the canonical mTORC1
substrates
,- (D1-D5) other rare tumours exhibit high level signal on both mTORC1 substrates simultaneously
→ would you have reached these conclusions using bulk methods?
In the last ten years we have gone from trying to
understand organs to trying to understand single cells to
understanding different omics within a cell
Especially transomics – correlating different layers of
omics within a cell – is becoming more and more
essential to understand single cell behaviour
Cytometry: technology that measures properties of single cells
Parameters measured:
- fluorescence
- light scatter
- other properties of cells and particles depending on how the composition is configured
Cytometry provides correlated data that links different population profiles
What is a flow cytometer: a system for measuring and then analysing the signals that result as particles flow in
a liquid stream through a beam of light
A flow cytometer does not always deal with cells; also with chromosomes, vesicles, latex beads, or any particle
that can be suspended in a fluid → you can only measure things in suspension, some particles more difficulty
because they prefer to be aggregated
Early flow cytometers were developed with the purpose of sorting particles based on their fluorescence (FACS
– fluorescence-activated cell sorting), although many cytometers today are in fact not capable of sorting, they
are still referred to as FACS
Applications of cytometry:
- enumerate particles/cells in suspension
- determine ‘’biologicals’’ from ‘’non-biologicals’’
- separate ‘’live’’ from ‘’dead’’ particles
- evaluate 105 to 107 particles in less than a minute
- measure particle-scatter, autofluorescence, or fluorescence associated to other reagents (antibodies and non-
antibodies)
- sort single particles/cells for subsequent analysis
- but also:
obtain images
measure rare metals
measure the entire spectrum of light
Benefits of high dimensional microscopy:
- multiplexed tissue image → spatial composition analysis → epitope presence
- single cell segmentation → single cell analysis → comprehensive cell states
- cell neighbourhoods → neighbourhood analysis → neighbourhood properties
Ghost cytometry – interesting article to read
,Oil droplet encapsulation:
- input = single cells in suspension + 10x gel beads and
reagents
- output = digital gene expression profiles from every
partitioned cell
The gel beads contain barcoded cDNA → transcript from
single cells of interest can be tagged with molecular
identifiers corresponding to the different beads
→ count how many transcripts there are per identifier and
to which cell they belong → how many transcripts in each
cell type etc.
Prior to lipid encapsulation, cells can get incubated with antibodies that are also marked with a DNA barcode
Both information at the transcript level and protein expression level. However, there is difficulty to find the
right dilution of antibodies that you expose to the cell, overdoing can result in nonspecific staining, not adding
enough can result in low binding (not enough antibody to measure signal) leading to wrong conclusions →
need to do titrations to determine this
The beginnings of cytometry: the flow cytometers that we use today still have the same
elements as the first cytometers that were developed. Important elements to consider:
- the cells, what kind of chemistry are we using to interrogate different aspects of the
cells
- what kind of fluidics are we using → is going to determine how fast and accurately the
cells can be measured
- the illumination/optics, ability to adequately get data, illumination that is being used
(488 nm,594 nm etc.) needs to be in sync with the laser that is being used
- detection and electronics, will count the amount of photons → output, huge table
containing columns and rows with every row a cell and every column a colour
, Three types:
- conventional flow cytometry and sorting
chemistry: based on fluorescence/fluorochromes
illumination: lasers
fluidics: hydrodynamic/acoustic
detection: PMT, APD, CCD
- mass cytometry
chemistry: rare metals that are conjugated to ABs, bind DNA/proteins
illumination: ICP
fluidics: nebulizer
detection: TOF-MS
often used with fixed cells, then toxicity is no problem, however with sorting live/dead cells
platinum can be used and if you incubate cells with platinum for too long it is toxic
→ important consideration is the toxicity of some metals, usually conjugated to antibodies toxicity
is not a problem
- single cell sequencing
chemistry: DNA-barcoded Ab
illumination: -
fluidics: microfluidics
detection: sequencing
Cytometry modalities
- conventional cytometry
acoustic focusing – helps you put the cells in a stream using acoustic forces to create
vibration patterns that will push the cells towards the middle of the stream
hydrodynamic focusing – uses water to push the cells towards the middle of the stream
PMT (photomultiplier tubes – fluorescence detection) /APD/CCD
- spectral cytometry
hybrid PMT/spectral
full spectral
- imaging flow cytometry – a lot of technical difficulties to achieve imaging whilst analysing
- mass cytometry – need to prepare millions of cells because the yield of detection is quite low
- sorting
cuvette – measuring through a cuvette, the light suffers no decay
jet-in-air – laser has to go through optical glass fibre – change in matter results in decay of light
cartridge
beads – e.g. magnetic beads
→ less sensitivity in the last three ones but decreased changes in pressure that results in less stress
for the cells, not as altered measurements
Some approaches are designed to be conventional cytometry but can also be applied for spectral cytometry
IMMUNOTHERAPY MODALITIES 7-9-2023
The pillars of cancer care:
- surgery
- radiotherapy
- cytotoxic chemotherapy
- molecularly targeted therapy
- immunotherapy
INTRODUCTION, SINGLE CELL MODALITIES 5-9-2023
Goal of the course: becoming familiar quantification
and monitoring of immune responses
Paper discussed during WG and presentations are not
part of the exam, open-ended exam questions
If you want to reach course coordinator use
amsterdamumc email
Immunotherapy: a type of treatment in which the
immune system is modulated (enhanced or
suppressed)
with the ultimate goal of curing/alleviating a disease
The immune system has tremendous heterogeneity, how do different cell types contribute in different tissues
→ needs to be studied on a single cell level to completely understand
Biological systems are composed of heterogeneous cell types in which minority cell populations play important
roles:
- in many tumours, only a small infraction (typically <1%) of molecularly distinct cells called ‘Cancer Stem Cells’
have the capacity for self-renewal and tumorigenesis → understanding these cells has an enormous
therapeutic potential
- a minority of tissue-specific stem cell populations are responsible for tissue development, regeneration and
repair
- the immune system is a classic example for the need of single cell technologies, because its dynamic cellular
heterogeneity is essential for fighting off the variety of attacking pathogens
Bulk techniques:
- RNA sequencing
- PCR
- Western blotting
- proteomics
Single cell techniques:
- flow cytometry
- single cell RNA sequencing
- microscopy – observing single cells using segmentation, masks
Understanding tissue heterogeneity:
mTOR targets phospho-ribosomal protein S6 and phosphor-4E-BP1 exhibit substantial
cellular exclusivity in colorectal cancer speciments, with rare co-expression
- rows (A-C) show different patterns of mutually exclusive p4E-BP1 (red) and pS6
(green) signals
- (A1-A3) many specimens exhibit a strong signal for each phosphorylation event in
exclusive tumour cells
- (A4-C2) another notable pattern exhibits substantial p4E-BP1 expression in tumour
cells, and a high level pS6 signal in stromal cells
- (C3-C5) rare tumours exhibit high level signal in only one of the canonical mTORC1
substrates
,- (D1-D5) other rare tumours exhibit high level signal on both mTORC1 substrates simultaneously
→ would you have reached these conclusions using bulk methods?
In the last ten years we have gone from trying to
understand organs to trying to understand single cells to
understanding different omics within a cell
Especially transomics – correlating different layers of
omics within a cell – is becoming more and more
essential to understand single cell behaviour
Cytometry: technology that measures properties of single cells
Parameters measured:
- fluorescence
- light scatter
- other properties of cells and particles depending on how the composition is configured
Cytometry provides correlated data that links different population profiles
What is a flow cytometer: a system for measuring and then analysing the signals that result as particles flow in
a liquid stream through a beam of light
A flow cytometer does not always deal with cells; also with chromosomes, vesicles, latex beads, or any particle
that can be suspended in a fluid → you can only measure things in suspension, some particles more difficulty
because they prefer to be aggregated
Early flow cytometers were developed with the purpose of sorting particles based on their fluorescence (FACS
– fluorescence-activated cell sorting), although many cytometers today are in fact not capable of sorting, they
are still referred to as FACS
Applications of cytometry:
- enumerate particles/cells in suspension
- determine ‘’biologicals’’ from ‘’non-biologicals’’
- separate ‘’live’’ from ‘’dead’’ particles
- evaluate 105 to 107 particles in less than a minute
- measure particle-scatter, autofluorescence, or fluorescence associated to other reagents (antibodies and non-
antibodies)
- sort single particles/cells for subsequent analysis
- but also:
obtain images
measure rare metals
measure the entire spectrum of light
Benefits of high dimensional microscopy:
- multiplexed tissue image → spatial composition analysis → epitope presence
- single cell segmentation → single cell analysis → comprehensive cell states
- cell neighbourhoods → neighbourhood analysis → neighbourhood properties
Ghost cytometry – interesting article to read
,Oil droplet encapsulation:
- input = single cells in suspension + 10x gel beads and
reagents
- output = digital gene expression profiles from every
partitioned cell
The gel beads contain barcoded cDNA → transcript from
single cells of interest can be tagged with molecular
identifiers corresponding to the different beads
→ count how many transcripts there are per identifier and
to which cell they belong → how many transcripts in each
cell type etc.
Prior to lipid encapsulation, cells can get incubated with antibodies that are also marked with a DNA barcode
Both information at the transcript level and protein expression level. However, there is difficulty to find the
right dilution of antibodies that you expose to the cell, overdoing can result in nonspecific staining, not adding
enough can result in low binding (not enough antibody to measure signal) leading to wrong conclusions →
need to do titrations to determine this
The beginnings of cytometry: the flow cytometers that we use today still have the same
elements as the first cytometers that were developed. Important elements to consider:
- the cells, what kind of chemistry are we using to interrogate different aspects of the
cells
- what kind of fluidics are we using → is going to determine how fast and accurately the
cells can be measured
- the illumination/optics, ability to adequately get data, illumination that is being used
(488 nm,594 nm etc.) needs to be in sync with the laser that is being used
- detection and electronics, will count the amount of photons → output, huge table
containing columns and rows with every row a cell and every column a colour
, Three types:
- conventional flow cytometry and sorting
chemistry: based on fluorescence/fluorochromes
illumination: lasers
fluidics: hydrodynamic/acoustic
detection: PMT, APD, CCD
- mass cytometry
chemistry: rare metals that are conjugated to ABs, bind DNA/proteins
illumination: ICP
fluidics: nebulizer
detection: TOF-MS
often used with fixed cells, then toxicity is no problem, however with sorting live/dead cells
platinum can be used and if you incubate cells with platinum for too long it is toxic
→ important consideration is the toxicity of some metals, usually conjugated to antibodies toxicity
is not a problem
- single cell sequencing
chemistry: DNA-barcoded Ab
illumination: -
fluidics: microfluidics
detection: sequencing
Cytometry modalities
- conventional cytometry
acoustic focusing – helps you put the cells in a stream using acoustic forces to create
vibration patterns that will push the cells towards the middle of the stream
hydrodynamic focusing – uses water to push the cells towards the middle of the stream
PMT (photomultiplier tubes – fluorescence detection) /APD/CCD
- spectral cytometry
hybrid PMT/spectral
full spectral
- imaging flow cytometry – a lot of technical difficulties to achieve imaging whilst analysing
- mass cytometry – need to prepare millions of cells because the yield of detection is quite low
- sorting
cuvette – measuring through a cuvette, the light suffers no decay
jet-in-air – laser has to go through optical glass fibre – change in matter results in decay of light
cartridge
beads – e.g. magnetic beads
→ less sensitivity in the last three ones but decreased changes in pressure that results in less stress
for the cells, not as altered measurements
Some approaches are designed to be conventional cytometry but can also be applied for spectral cytometry
IMMUNOTHERAPY MODALITIES 7-9-2023
The pillars of cancer care:
- surgery
- radiotherapy
- cytotoxic chemotherapy
- molecularly targeted therapy
- immunotherapy
